Compositions and methods for neural differentiation of embryonic stem cells

ABSTRACT

The present invention provides compositions and methods for human neural cell production. More particularly, the present invention provides cellular differentiation methods employing an essentially serum free MEDII conditioned medium for the generation of human neural cells from pluripotent and multipotent human stem cells.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to human neural cells and todifferentiated or partially differentiated cells derived therefrom. Thepresent invention also relates to methods of producing, differentiatingand culturing the cells of the invention, and to uses thereof.

2. Background Art

In the human and in other mammals, formation of the blastocyst,including development of inner cell mass (ICM) cells and theirprogression to pluripotent cells of the primitive ectoderm, andsubsequent differentiation to form the embryonic germ layers anddifferentiated cells, follow a similar developmental process.

Embryonic stem (ES) cells represent a powerful model system for theinvestigation of mechanisms underlying pluripotent cell biology anddifferentiation within the early embryo, as well as providingopportunities for genetic manipulation of mammals and resultantcommercial, medical and agricultural applications. Furthermore,appropriate proliferation and differentiation of ES cells can be used togenerate an unlimited source of cells suited to transplantation fortreatment of diseases that result from cell damage or dysfunction. Otherpluripotent cells and cell lines including early primitive ectoderm-like(EPL) cells as described in International Patent Application WO99/53021, in vivo or in vitro derived ICM/epiblast, in vivo or in vitroderived primitive ectoderm, primordial germ cells (EG cells),teratocarcinoma cells (EC cells), and pluripotent cells derived bydedifferentiation or by nuclear transfer will share some or all of theseproperties and applications.

The successful isolation, long term clonal maintenance, geneticmanipulation and germ-line transmission of pluripotent cells fromspecies other than rodents has generally been difficult and the reasonsfor this are unknown. International Patent Application WO 97/32033 andU.S. Pat. No. 5,453,357 describe pluripotent cells including cells fromspecies other than rodents. Human ES cells have been described inInternational Patent Application WO 96/23362, and in U.S. Pat. No.5,843,780, and human EG cells have been described in InternationalPatent Application WO 98/43679.

The ability to tightly control differentiation or form homogeneouspopulations of partially differentiated or terminally differentiatedcells by differentiation in vitro of pluripotent cells has provedproblematic. Current approaches involve the formation of embryoid bodiesfrom pluripotent cells in a manner that is not controlled and does notresult in homogeneous populations. Mixed cell populations such as thosein embryoid bodies of this type are generally unlikely to be suitablefor therapeutic or commercial use.

Uncontrolled differentiation produces mixtures of pluripotent stem cellsand partially differentiated stem/progenitor cells corresponding tovarious cell lineages. When these ES-derived cell mixtures are graftedinto a recipient tissue the contaminating pluripotent stem cellsproliferate and differentiate to form tumors, while the partiallydifferentiated stem and progenitor cells can further differentiate toform a mixture of inappropriate and undesired cell types. It is wellknown from studies in animal models that tumors originating fromcontaminating pluripotent cells can cause catastrophic tissue damage anddeath. In addition, pluripotent cells contaminating a cell transplantcan generate various inappropriate stem cell, progenitor cell anddifferentiated cell types in the donor without forming a tumor. Thesecontaminating cell types can lead to the formation of inappropriatetissues within a cell transplant. These outcomes cannot be tolerated forclinical applications in humans. Therefore, uncontrolled ES celldifferentiation makes the clinical use of ES-derived cells in human celltherapies impossible.

Selection procedures have been used to obtain cell populations enrichedin neural cells from embryoid bodies. These include manipulation ofculture conditions to select for neural cells (Okabe et al., 1996 Dev.Biol. 176:300-312; and Tropepe et al., 2001 Neuron 30:65-78; O'Shea,2002 Meth. in Mol. Biol. 198, 3-14), and genetic modification of EScells to allow selection of neural cells by antibiotic resistance (Li etal., 1998 Current Biol. 8:971-974). Previously, one research group hasdemonstrated efficient differentiation of mouse and primate ES cells toTH⁺ neurons following co-culture with the PA6 stromal cell line, butthis technique is not likely to be useful for cell therapy applicationsas it introduces xenograft issues associated with exposure to non-humancell lines and removal of potential PA6 cell contamination in subsequentcultures (Kawasaki et al., 2000 Neuron 28, 31-40; Kawasaki et al., 2002Proc. Natl. Acad. Sci. USA, 99(3): 1580-1585). Furthermore, the PA6differentiation procedure generated non-neural terminally differentiatedcell types, such as retinal epithelial cells, reducing the usefulness ofthe cell cultures for cell therapy. In addition, McKay has demonstratedefficient differentiation of mouse ES cells to TH+ neurons, but thisdifferentiation required over-expression of the Nurr-1 transcriptionfactor in combination with exposure to Sonic Hedgehog and FGF8 (Kim etal., Nature 2002 418(6893):50-6). Furthermore, the McKay protocolinvolves a complex, five stage differentiation method fordifferentiation of mouse ES cells to neurons.

Another research group differentiated human ES cell derived embryoidbodies in 20% serum containing medium for 4 days followed by plating andselection/expansion of neural cell types in medium containing B27 and N2supplements (serum free), EGF, FGF-2, PDGF-AA, and IGF-1 (Carpenter etal., 2001 Exper. Neuro. 172, 383-397). Carpenter et al. showed thatneural progenitors could be enriched from this culture system by cellsorting or immunopanning using antibodies directed against polysialatedNCAM or the cell surface molecule recognized by the A2B5 monoclonalantibody.

Another emerging approach to isolate neural cells is the use oftransgenes that express a marker such as green fluorescent protein (GFP)under the control of a lineage specific promoter. The transgene allowscell sorting of the neural cells that differentiate from the ES culturesafter retinoic acid and sonic hedgehog expression were used to inducethe formation of spinal cord motor neurons (Steven Goldman, NationalInstitutes of Health Symposium, NIH Research: Recent Progress and FuturePromise of Human Embryonic Stem Cells, Jun. 12, 2003, abstract availableat stemcells.nih.gov/news/symposiumSpeakers.asp#7 as of Jul. 30, 2003).

In all of these procedures, the differentiation of pluripotent cells invitro does not involve biological molecules that direct differentiationin a controlled manner, that parallels or mimics the developmentalstages of neural differentiation that occur in vivo. Similarly, inexperiments examining neural differentiation from human ES cells, thereis no way to control the neural differentiation, and the methods merelyallow for the passive development of neural cell types (see Zhang etal., 2001 Nature Biotech 19(12): 1129-1133, and Reubinoff et al., 2001Nature Biotech 19(12); 1134-40). Hence homogeneous, synchronouspopulations of neural cells with unrestricted neural differentiationcapability are not produced, restricting the ability to deriveessentially homogeneous populations of partially differentiated ordifferentiated neural cells.

Efficient neural differentiation of mouse embryonic stem cells inmonolayer culture has recently been reported (Ying et al., 2003 NatureBiotechnology, 21:183-186). This previous study shows that adherentmouse ES cells can differentiate into neural cell types in a serum-freeminimal medium. In contrast to the work described herein, the methoddescribed by Ying et al. produces neuronal cultures containing manyGABAergic neurons and very few tyrosine hydroxylase expressing neurons.In addition the methods of Ying et al. are dependent on monolayerculture of the mouse ES cells.

Chemical inducers such as retinoic acid have also been used to formneural lineages from a variety of pluripotent cells including ES cells(Bain et al., 1995 Dev. Biol. 168:342-357; Strubing et al., 1995 Mech.Dev. 53, 275-287; Fraichard et al., 1995 J. Cell Sci. 108, 3181-3188;Schuldiner et al., 2001 Brain Res. 913, 201-205). However, the route ofretinoic acid-induced neural differentiation has not been wellcharacterized, and the repertoire of neural cell types produced appearsto be generally restricted to ventral somatic motor, branchiomotor orvisceromotor neurons (Renoncourt et al., 1998 Mech. Dev. 79:185-197).

Manually passaged HESC colonies are typically comprised of tightlypacked, multilayered, undifferentiated HESCs, and variable levels ofcells undergoing early differentiation. When present, thesedifferentiating cells are observed on the edges of HESC colonies and areconsidered to be an indicator that the maintenance of theundifferentiated state of the colony is beginning to be compromised.This is undesirable as the presence of differentiating cells is likelyto have a negative influence on maintaining the undifferentiated stateof the remaining HESC, as the differentiating cells can produce factorsthat influence cellular differentiation. Furthermore, the presence ofdifferentiated cells is likely to add randomness to differentiationprocedures due to the stochastic presence of these cells and thedifferentiation signals or factors that they produce. Due to the threedimensional nature of the manually passaged HESC cultures,differentiating cells are also likely to be present in regions of thecolonies where they cannot be detected or distinguished morphologically.As shown by Henderson et al. (Stem Cells, 2002, 20:329-337), SSEA3 orSSEA1 magnetic bead based sorting of cells confirms the likelihood ofdifferent cell populations within a culture akin to manually passagedHESC cultures. There is therefore a need to develop methods to passageHESCs that result in more uniform populations of undifferentiated orpartially undifferentiated cells, and that are not based onmorphological distinctions.

Previous publications report the transplantation of ES-derived neuralcells into the ventricles of the fetal or newborn rat or mouse brainwithout the formation of tumors (Brustle et al., 1997 PNAS 94,14809-14814, Zhang et al., 2001 Nature Biotech 19, 1129-1133). Althoughsome of the cells in these studies do integrate into the host brain,many of the cells in the transplants form neural tube like structureswithin the lumen of the brain ventricle. Therefore, these previousstudies do not lead to methods that can be readily applied to human celltherapy. Note that Reubinoff et al. (2001 Nature Biotech 19, 1134) alsoinjected into the ventricles of newborn mice but did not reportintraventricular masses of neural cells, omitting any mention of thepresence or absence of such masses.

Neural stem cells and precursor cells have been derived from fetal brainand adult primary central nervous system tissue in a number of species,including rodent and human (e.g., see U.S. Pat. No. 5,753,506 (Johe),U.S. Pat. No. 5,766,948 (Gage), U.S. Pat. No. 5,589,376 (Anderson andStemple), U.S. Pat. No. 5,851,832 (Weiss et al.), U.S. Pat. No.5,958,767 (Snyder et al.) and U.S. Pat. No. 5,968,829 (Carpenter)).However, each of these disclosures fails to describe a predominantlyhomogeneous population of neural stem cells able to differentiate intoall neural cell types of the central and peripheral nervous systems,and/or essentially homogeneous populations of partially differentiatedor terminally differentiated neural cells derived from neural stem cellsby controlled differentiation. Furthermore, it is not clear whethercells derived from primary fetal or adult tissue can be expandedsufficiently to meet potential cell and gene therapy demands. Neuralstem cells derived from fetal or adult brain are established andexpanded after the cells have committed to the neural lineage and insome cases after the cells have committed to neural sublineages.Therefore these cells do not provide the opportunity to manipulate theearly differentiation processes that occur prior to neural commitment.Pluripotent stem cells provide access to these earliest stages ofmammalian cellular differentiation opening additional options for cellexpansion and directed development of the cells into desired lineages.

In summary, it has not been possible to control the differentiation ofpluripotent cells in vitro, to provide homogeneous, synchronouspopulations of neural cells with unrestricted neural differentiationcapacity. Similarly, methods have not been developed for the derivationof neural cells from pluripotent cells in a manner that parallels theirformation during embryogenesis. In addition, current methods have reliedupon the expression of foreign genes to drive neural differentiation ofpluripotent stem cells (Kim et al., 2002 Nature 418:50-56). Theselimitations have restricted the ability to form essentially homogeneous,synchronous populations of partially differentiated and terminallydifferentiated neural cells in vitro, and have restricted their furtherdevelopment for therapeutic and commercial applications.

There is a need, therefore, to identify methods and compositions for theproduction of a population of cells enriched in neural stem cells andthe products of their further differentiation, and in particular, humanneural cells and their products.

SUMMARY OF THE INVENTION

It is an object of the present invention to overcome, or at leastalleviate, one or more of the difficulties or deficiencies associatedwith the prior art. In that regard, the present invention particularlyprovides a method of producing a human neural cell that includes thesteps of: a) providing a pluripotent human cell; b) culturing thepluripotent human cell with an essentially serum free medium to form anembryoid body, and c) culturing the embryoid body with an essentiallyserum free cell differentiation environment until the human neural cellis produced. The present invention also provides a method of enriching ahuman cell culture for neural cells including the following steps: a)providing a pluripotent human cell culture; b) culturing the pluripotenthuman cell culture with an essentially serum free MEDII conditionedmedium to form an embryoid body; and c) culturing the embryoid body withan essentially serum free cell differentiation environment until a humancell culture enriched in neural cells is produced. In certain preferredembodiments, the essentially serum free medium is a MEDII conditionedmedium.

The MEDII conditioned medium described herein is preferably a Hep G2conditioned medium that contains a bioactive component selected from thegroup consisting of: a large molecular weight extracellular matrixprotein; a low molecular weight component comprising proline; abiologically active fragment of any of the aforementioned proteins orcomponents; and an analog of any of the aforementioned proteins orcomponents. The pluripotent human cell of the present invention can beselected from, but is not limited to, the group consisting of a humanembryonic stem cell; a human ICM/epiblast cell; a human EPL cell; ahuman primitive ectoderm cell; a human primordial germ cell; and a humanteratocarcinoma cell.

In other embodiments of the present invention, the methods describedabove further include the steps of dispersing the embryoid body to anessentially single cell suspension; culturing the essentially singlecell suspension in a suspension culture and forming a second embryoidbody by contacting the essentially single cell suspension with a secondessentially serum free medium wherein the second essentially serum freemedium is a MEDII conditioned medium; and contacting the second embryoidbody with an essentially serum free cell differentiation environmentuntil the human neural cell is produced, or the human cell cultureenriched in neural cells is produced.

The invention provides a composition comprising a culture of neuralcells, wherein the neural cells are preferably neural progenitor cells.The neural progenitor cells are characterized by the expression ofnestin or vimentin, and their capacity to differentiate into cells ofthe neural lineage including neurons and glia. The neural cell typesproduced may include cells of the central or peripheral nervous system,including, but not limited to neurons, astrocytes, oligodendrocytes andSchwann cells. Neuron cell types produced in these cultures may expressone or more neurotransmitter phentotypes. These include GABAergicneurons that express glutamate decarboxylase (GAD) or vesicularinhibitory amino acid transporter/vesicular gaba transporter(Viaat/Vgat); cholinergic neurons that express choline acetyltransferase(ChAT/CAT) or vesicular acetylcholine transporter (VAChT); glutamatergicneurons that express the vesicular glutamate transporter; glycinergicneurons that express the vesicular inhibitory amino acid transporter(Viaat/Vgat), noradrenergic neurons that express the norepinephrinetransporter (NET); adrenergic neurons that express phenylmethanolamineN-methyl transferase (PNMT); serotonergic neurons that expresstryptophan hydroxylase (TrH) or serotonin transporter (SERT); orhistaminergic neurons that express histidine decarboxylase (HDC).

The invention further provides a composition comprising a culture ofneural cells comprising neural cells derived in vitro from a pluripotentor multipotent cell. In preferred embodiments, these neural cells arecapable of expressing one or more of the detectable markers for tyrosinehydroxylase (TH), vesicular monamine transporter (VMAT) dopaminetransporter (DAT), and aromatic amino acid decarboxylase (AADC, alsoknown as dopa decarboxylase). Preferably, the neural cell cultureexpresses all of the detectable markers for TH, VMAT, DAT, and AADC.Such a neural cell culture was not previously available and can beproduced by the methods described herein or by other methods laterdeveloped.

The invention further provides methods of producing a partiallydifferentiated pluripotent cell comprising culturing pluripotent cellson a layer of fresh feeder cells, wherein the fresh feeder cells areless than 2 days old, thereby inducing formation of a moredifferentiated pluripotent cell. In preferred embodiments, the freshfeeder cells are less than one day old, more preferably less than 12hours old, or more preferably less than 6 hours old. In preferredembodiments, the more differentiated pluripotent cell is obtained fromthe central, or crater, region of the colony of pluripotent cells. Insome embodiments, the more differentiated pluripotent cell expressesless Oct4 marker than an embryonic stem cell. The invention furtherprovides a composition comprising these more differentiated pluripotentcells.

The invention further provides a method of treating a patient with aneural disease, comprising a step of administering to the patient atherapeutically effective amount of the neural cell or cell cultureenriched in neural cells produced using the methods of the presentinvention.

The invention further provides for the human pluripotent cells, thehuman neural precursor cells and human neural cells produced using themethods of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show derived serum free embryoid bodies. Human embryonicstem cell colonies were dissected into uniform sized pieces and culturedin suspension. Solid serum free embryoid bodies (FIG. 1A) weredistinguished from structured serum free embryoid bodies (FIG. 1B) afterone week.

FIGS. 2A-2B show neural differentiation of serum free embryoid bodies.FIG. 2A shows proliferation of neural progenitor rosettes observed inserum free embryoid bodies seeded in the presence of 50% MEDII. FIG. 2Bshows neurite network differentiation observed in serum free embryoidbodies seeded in the presence of 50% MEDII.

FIGS. 3A-3C show serum free embryoid bodies containing neuralprecursors. FIG. 3A shows phase contrast micrograph of seeded structured(left) and solid (right) serum free embryoid bodies. Greyscale images offluorescent stainings with anti-Sox1 is shown in FIG. 3B and anti-Map2antibodies is shown in FIG. 3C. Solid serum free embryoid bodies containa high proportion of neural precursors (Sox1⁺ cells) and rare neurites(Map2⁺). Structured serum free embryoid bodies contain rosettes ofsemi-differentiated neural precursors (Sox1⁺/Map2⁺) and neurites(Map2⁺).

FIGS. 4A-4B show Map2 and nestin expression in rosettes. Radial Map2⁺and nestin+ expression indicates a semi-differentiated progenitor/youngneuron.

FIGS. 5A-5C show Sox1, Map2, and DAPI expression in serum free embryoidbody differentiation. Map2 expression in FIG. 5A indicatessemi-differentiated neurons (radial pattern) and differentiated neurons(network, dendritic stain), Sox1 expression in FIG. 5B indicates neuralprecursors, and DAPI in FIG. 5C is a DNA stain highlighting all nucleiin the field. Virtually all nuclei in the rosette are double stained forSox1/DAPI, indicating a relatively pure population of neural progenitors(Sox1⁺) and/or semi-differentiated neural progenitors (Sox1⁺/Map2⁺).

FIGS. 6A-D show Sox1, nestin and Map2 expression in serum free embryoidbody differentiation. A phase contrast micrograph of a region of adifferentiating serum free embryoid body is shown in FIG. 6A. Rosette,neuron and other cell types are present. Greyscale images of fluorescentstainings with anti-nestin in FIG. 6B, anti-Sox1 in FIG. 6C andanti-Map2 in FIG. 6D are shown. Observed cell types are: neuralprogenitors (Nestin⁺/Sox1⁺) and/or semi-differentiated neurons(Nestin⁺/Sox1⁺/radial Map2⁺); differentiated neurons (Map2⁺), and otherprogenitor cell types (Nestin⁺).

FIG. 7 shows a network of anti-Tyrosine Hydroxylase stained neurons in acrater colony derived sfEBMs plated onto a polyornithine/laminin matrix

FIGS. 8A-8B show co-expression of Tyrosine Hydroxylase (FIG. 8A) andMap2 (FIG. 8B) shown by fluorescent immunostaining of neurons in acrater derived serum free embryoid body plated onto apolyornithine/laminin matrix.

FIGS. 9A-9B show crater derived serum free embryoid bodies at day 7 insuspension in (FIG. 9A) DMEM F12/N2/FGF-2 and (FIG. 9B) DMEMF12/N2/FGF-2/50% MEDII.

FIGS. 10A-D show expression of Oct4 protein in HESCs and serum freeembryoid bodies. FIG. 10A shows high levels of Oct4 expression in atypical manually passaged HESC colony, with distinct nuclear expressionin undifferentiated ES cells and no Oct4 in the unstained feeder layersurrounding the HESC colony. FIG. 10B shows a typical manually passagedHESC crater colony, showing high levels of Oct4 expression in themultilayered ring of undifferentiated cells surrounding the monolayercrater cells that express a low level of Oct4. Differentiating cells atthe edge of the colony also express a low level of Oct4. FIG. 10C showsthe expression of Oct4 in a seeded essentially serum free embryoid body,representative of what is seen when sfEBMs are derived from domed HESCsor monolayer crater cells. Regions of high level Oct4 expression persistand are indicative of residual nests of pluripotent cells maintained bylocal cell-cell signaling events. Neural rosettes in the same field areindicated as radially organized circles of nuclei by DAPI staining (FIG.10D) and these neural precursor cells only express low levels of Oct4.

FIGS. 11A-L show immunostaining of SSEA4 selected trypsin passagedcells. FIGS. 11A and B show Oct4 and DAPI staining, respectively; FIGS.11C and D show SSEA1 and DAPI staining, respectively; FIGS. 11E and Fshow SSEA3 and DAPI staining, respectively; Figs. G and H show SSEA4 andDAPI staining, respectively, FIGS. 11I and J show Tra-1-60 and DAPIstaining, respectively; and FIGS. 11K and L show Tra-1-81 and DAPIstaining respectively.

FIGS. 12A-D show Nestin expression in manually passaged and SSEA4selected trypsin passaged cells. FIGS. 12A and B show Nestin and DAPIstaining of manually passaged HESCs, respectively. The edge of a HESCcolony is shown, showing that multilayered cells toward the center ofthe colony do not exhibit nestin expression (indicated by the dot in thelower right corner), while nestin expressing cells encircle the colony(indicated by the arrowhead), which are in turn surrounded by an outerring of differentiating nestin+ cells (top left corner, indicated by thearrow). Figs. C and D show Nestin and DAPI staining of SSEA4 selectedtrypsin passaged HESCs, respectively. A substantially uniformdistribution of nestin is exhibited.

FIGS. 13A-D show enhanced neural differentiation of SSEA4 selectedtrypsin passaged HESCs in response to MEDII. Serum free embryoid bodieswere derived, exposed to 10 μM S18 from day 13 to day 17, seeded at day18 and fixed for immunostaining at day 23. A and B show THimmunostaining and DAPI staining, respectively, of serum free embryoidbodies grown in FGF2. The proportion of TH⁺ cells and distribution ofthe network of the dopaminergic neural projections was considerablyenhanced over what had previously been observed with serum free embryoidbodies derived from manually passaged HESCs. Up to 30-70% by area of thesfEBs contained TH⁺ neurons, as opposed to less than ˜20% for craterderived sfEBMs. Significant regions of the seeded embryoid bodies didnot contain neurons. C and D show TH immunostaining and DAPI staining,respectively, of serum free embryoid bodies grown in FGF2/MEDII. A veryhigh proportion of the culture, typically >90% of the area of a seededsfEBM piece, consisted of TH⁺ neurons and the differentiation of thesecells was enhanced, as they exhibited far more developed neuralprocesses. Non-neural regions of the culture were significantly reduced.The proportion of neural rosettes appeared to be far greater in culturesexposed to MEDII.

FIGS. 14A-F show high efficiency dopaminergic differentiation. SSEA4selected trypsin passaged HESCs were differentiated in response to MEDIIto generate a very high proportion of TH⁺ neurons. Serum free embryoidbodies were derived, exposed to 10 μM S18 from day 13 to day 17, seededat day 18 and fixed for immunostaining at day 23. A and B showβIII-Tubulin and DAPI staining, respectively, of a seeded sfEBM. Theboxes mark the regions shown at increased magnification in C-F. C and Eshow an increased magnification of the TH immunostaining, and D and Fshow an increased magnification of the βIII-Tubulin immunostaining. Avery high proportion, typically 90% or greater of the neurons expressTH.

FIGS. 15A-B show a comparison of TH⁺ and Hoffman optics images of neuralextensions in a region of a serum free embryoid body grown in 4 ng/mlFGF2. Serum free embryoid bodies were derived, exposed to 10 μM S18 fromday 13 to day 17, seeded at day 18 and fixed for immunostaining at day23. A very high proportion of neurons express TH.

FIGS. 16A-D show expression of TH and VMAT in sfEBM cultures. sfEBMswere derived, exposed to 10 μM S18 from day 13 to day 17, seeded at day18 and fixed for immunostaining at day 23. A and C show VMAT expressionat 40× and 20× magnification respectively. B and D show TH expression at40× and 20× magnification respectively. TH⁺/VMAT⁻, TH⁻/VMAT⁺ andTH⁺/VMAT⁺ cells could be observed.

FIGS. 17A-B illustrate the dopamine release assay. FIG. 17A is aschematic representation of the purification, modification andcompetitive enzyme linked immunoassay. Dopamine (D) is released fromcultured neurons by depolarization with KCl, D is then is purified witha cis-diol affinity resin and acylated to N-acyldopamine (D^(a)). D^(a)remains in suspension and is modified to N-acyl-3-Methoxytyamine (m),which competes with solid phase D for a limited number of anti-dopamineantibody binding sites. Free antigen and antibody are removed bywashing, and antibody bound to solid phase D is detected with asecondary antibody-peroxidase conjugate. There is an inverse correlationbetween the amount of D in the samples and detected signal. The amountof D in the sample is established from a standard curve. FIG. 17B showsa determination of dopamine released from sfEBM samples, which had beenderived, seeded to polyornithine/laminin coated slides, at day 25 andcultured to day 30 prior to depolarization. The cultures releasedapproximately 2650 pg/ml of dopamine into the depolarizing medium (dotand vertical line). This value was within the range of the standardcurve (dots representing 0, 150, 600, 2400, 9600, 38400 pg/ml dopamine)and fell between two unknown control samples from the kit (arrows).

FIGS. 18A-E show the neural differentiation of SSEA4 selected bulkpassaged cells cultured as serum free embryoid bodies in FGF2 andProline. sfEBP were derived and cultured for 10 or 17 days, and seededto polyornithine/laminin for 5 days. FIGS. 18A, and B show seeded sfEBPsat day 15 stained with DAPI and anti-βIII-Tubulin, respectively, at 10×magnification. FIGS. 18C, D, and E show seeded sfEBPs at day 22 stainedwith DAPI, anti-βIII-Tubulin and anti-TH, respectively, at 40×magnification.

FIG. 19 shows neural differentiation of SSEA4 selected bulk passagedcells cultured as serum free embryoid bodies in minimal medium withoutFGF2, MEDII or L-Proline. Serum free embryoid bodies were seeded at day21, fixed at day 25 and immunostained with anti-βIII-Tubulin and imagedat 10× magnification.

FIGS. 20A-B show, whole mount immunostaining and confocal analysis of 50μM L-Proline sfEBP at day 27 after derivation. Different sfEBPs areshown in these images. FIG. 20A shows anti-βIII-Tubulin immunostaining,detected with an Alexa 488 labeled secondary antibody and 1 μm confocalsection at 40× magnification. Complex networks of βIII-Tubulin positiveneuronal extensions were detected. A non-staining neural rosette isindicated by the asterisk, and βIII-Tubulin positive cell bodies areindicated by arrowheads. FIG. 20B shows a DAPI stained sfEBP imaged at 1μm sections by a 2-Photon laser confocal at 40× magnification. A largeproportion of the sfEBP consists of the elongated, closely packed,radial, neural rosette nuclei. The two dashed ovals surround a rosetteand indicate its central proliferative core, where mitotic figures arelocalized within rosettes.

DETAILED DESCRIPTION OF THE INVENTION

Applicant has demonstrated that contacting pluripotent human cells suchas human ES cells, with an essentially serum free medium results in theformation of a human neural cell type with greater efficiency orfrequency. The invention provides a neural cell culture compositioncharacterized by a variety of properties, heretofore unavailable. Theinvention further provides a partially differentiated pluripotent cellby culturing pluripotent cells on a fresh feeder cell layer, and methodsof obtaining the same.

The present invention particularly provides a method of producing ahuman neural cell that includes the steps of: a) providing a pluripotentor multipotent human cell; and b) contacting the pluripotent ormultipotent human cell with an essentially serum free celldifferentiation environment until the human neural cell is produced. Thepresent invention additionally provides a method of enriching a humancell culture for neural cells comprising the steps of: a) providing apluripotent or multipotent human cell culture; and b) contacting thepluripotent or multipotent human cell culture with an essentially serumfree cell differentiation environment until a human cell cultureenriched in neural cells is produced.

In preferred embodiments of the above methods, an embryoid body isformed upon culturing the pluripotent or multipotent human cell or cellculture with an essentially serum free medium, and as a subsequent step,the cells from the embryoid body are contacted with an essentially serumfree cell differentiation environment until a human neural cell or humancell culture enriched in neural cells is produced. In preferredembodiments, the essentially serum free medium is a MEDII conditionedmedium as defined herein. In other embodiments, the embryoid body iscontacted with one or more differentiation medium or celldifferentiation environments after being removed from the essentiallyserum free cell differentiation environment until a human neural cell orhuman cell culture enriched in neural cells is produced, wherein eachmedium or environment is appropriate to the cell types as they appearfrom the preceding cell type. It is to be understood that the absence ofthe term “differentiation” when describing a MEDII conditioned mediumdoes not indicate that the MEDII conditioned medium can not also beconsidered a “differentiation” medium. In certain embodiments, theessentially serum free medium preferably is also essentially LIF free.

In another preferred embodiment of the above method, an embryoid body isformed upon culturing the pluripotent or multipotent human cell or cellculture with an essentially serum free MEDII conditioned medium, thecells from the embryoid body are contacted with an essentially serumfree cell differentiation environment, the embryoid body is dispersed toan essentially single cell suspension, the single cell suspension iscultured in suspension culture, a second embryoid body is formed bycontacting the essentially single, cell suspension with a secondessentially serum free medium where the second essentially serum freemedium is a MEDII conditioned medium; and the second embryoid body is,contacted with an essentially serum free cell differentiationenvironment until the human neural cell is produced or human cellculture enriched in neural cells is produced. The second essentiallyserum free media can comprise DMEM/F12, FGF (e.g., FGF-2) and MEDIIconditioned media. In a preferred embodiment the MEDII conditioned mediacomprises 10-75% of the second essentially serum free media. Morepreferably, the MEDII conditioned media comprises 40-60% of the secondessentially serum free media, and most preferably the MEDII conditionedmedia comprises 50% of the second essentially serum free media. In apreferred embodiment the MEDII conditioned media comprises approximately1-30 ng/ml of FGF-2, more preferably approximately 2-10 ng-ml of FGF-2,and most preferably approximately 4 ng/ml of FGF-2.

Applicant has demonstrated that culturing human cell populationscomprising pluripotent human cells by selecting the cells with anantibody directed to a pluripotent cell marker, and/or passaging thecells with a protease treatment results in the formation of a humanpluripotent cell type that expresses cell markers characteristic ofhuman embryonic stem cells, and also expresses nestin in a substantiallyuniform manner. When these cells are cultured with MEDII, they formneural cells with greater homogeneity than observed in a pluripotenthuman cell population that is not cultured with MEDII. When these cellsare cultured with a minimal medium that optionally comprises proline,they form neural cells with greater homogeneity than observed in apluripotent human cell population that is not cultured with minimalmedium. This differentiation protocol has the capacity to be performedon a large scale, free of exposure to non-human cell types, to generatea high proportion of dopaminergic neurons, in the absence of residualpluripotent cells.

The invention provides a composition comprising a culture of neuralcells, wherein the neural cells are preferably neural progenitor cells.The neural progenitor cells are characterized by the expression ofnestin or vimentin, and their capacity to differentiate into cells ofthe neural lineage including neurons and glia. The neural cell typesproduced may include cells of the central or peripheral nervous system,including, but not limited to neurons, astrocytes, oligodendrocytes andSchwann cells. Neuron cell types produced in these cultures may expressone or more neurotransmitter phentotypes. These include GABAergicneurons that express glutamate decarboxylase (GAD) or vesicularinhibitory amino acid transporter/vesicular gaba transporter(Viaat/Vgat); cholinergic neurons that express choline acetyltransferase(ChAT/CAT) or vesicular acetylcholine transporter (VAChT); glutamatergicneurons that express the vesicular glutamate transporter; glycinergicneurons that express the vesicular inhibitory amino acid transporter(Viaat/Vgat), noradrenergic neurons that express the norepinephrinetransporter (NET); adrenergic neurons that express phenylmethanolamineN-methyl transferase (PNMT); serotonergic neurons that expresstryptophan hydroxylase (TrH) or serotonin transporter (SERT); orhistaminergic neurons that express histidine decarboxylase (HDC).

In one embodiment, the neural cell produced by culturing thedifferentiated pluripotent human cell is therapeutically transplantedinto the brain of a subject. The cell culture of the present inventionform teratomas at a greatly reduced frequency than if the culture wasnot treated with a serum free differentiation environment and/orpassaged using a protease treatment. In a preferred embodiment, the cellculture of the present invention does not induce the formation ofteratomas at a significant rate.

The present invention further provides a method of culturing a humanpluripotent cell, comprising the steps: a) selecting a human pluripotentcell using an anti-SSEA4 antibody; and b) maintaining a culture of thecell by passaging the cell using a protease treatment, wherein the cellsof the culture express SSEA3, SSEA4, Oct4, Tra-1-60, Tra-1-80, andexpress nestin substantially uniformly. As used herein, the term“substantially uniformly” refers to the expression pattern of a cellularmarker when a colony of cells is examined for expression of that marker.If there is “substantially uniform” expression of a marker, generallymost of the cells of the colony express the marker. For example, if thecenter of an HESC colony does not express a marker, but the marker isexpressed in most of the cells in the remainder of the colony, themarker is not expressed in a substantially uniform manner. Preferably,greater than 90% of the cells of a colony express the marker, morepreferably, greater than 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, of thecells of the colony express the marker, and still more preferably,greater than 99% of the cells of the colony express the marker.

In a preferred embodiment, the protease treatment comprises thesequential use of Collagenase and trypsin. In one embodiment,Collagenase is used at a concentration of from approximately 0.1 mg/mlto approximately 10 mg/ml, more preferably from a concentration of fromapproximately 0.5 mg/ml to approximately 5 mg/ml, and most preferably ata concentration of from approximately 1 mg/ml to 2 mg/ml. The inventioncontemplates that Collagenase may be used for approximately 1 minute to10 minutes, more preferably from approximately 2 minutes to 8 minutes,and most preferably for approximately 4 minutes to 6 minutes.

In another embodiment, trypsin is used at a concentration of fromapproximately 0.001% to 1%, more preferably at a concentration of fromapproximately 0.01% to 0.1%, and most preferably at a concentration ofapproximately 0.05%. The invention contemplates that trypsin may be usedfor approximately 1 second to 5 minutes, more preferably forapproximately 5 seconds to 2 minutes, more preferably for approximately10 seconds to 1 minute, and most preferably for approximately 30seconds.

In a further preferred embodiment, Collagenase is used at aconcentration of approximately 1 mg/ml for approximately 5 minutes, andtrypsin is used at a concentration of approximately 0.05% forapproximately 30 seconds.

The methods of the present invention further encompass providing a humancell culture enriched in neural cells, comprising the formation of anembryoid body that comprises a human pluripotent cell culture thatexpresses SSEA3, SSEA4, Oct4, Tra-1-60, Tra-1-80, and expresses nestinsubstantially uniformly. In one embodiment, the human pluripotent cellculture is provided using a protease passaging treatment. In anotherembodiment, the human pluripotent cell culture is provided usingantibody selection and protease passaging treatment. In anotherembodiment, the human pluripotent cell culture is provided usingantibody selection. In preferred embodiments of the invention, theantibody selection is performed using an anti-SSEA4 antibody. In afurther preferred embodiment, the protease passaging treatment comprisesthe use of Collagenase at a concentration of approximately 1 mg/ml forapproximately 5 minutes, and the subsequent use of trypsin at aconcentration of approximately 0.05% for approximately 30 seconds.

In a further preferred embodiment, the method of providing a human cellculture enriched in neural cells comprises the formation of an embryoidbody by culturing a human pluripotent cell culture with an essentiallyserum free medium. In a preferred embodiment, the essentially serum freemedium is a MEDII conditioned medium as defined herein. In anotherpreferred embodiment, the essentially serum free medium is a minimalmedium that optionally comprises proline.

In other embodiments of the present invention, it is not required thatan embryoid body is formed upon contacting the pluripotent ormultipotent human cell or cell culture with an essentially serum freeMEDII conditioned medium. In these embodiments, a pluripotent ormultipotent human cell or cell culture is contacted with a MEDIIconditioned medium, and as a subsequent step, the resultant cells can becontacted with an essentially serum free cell differentiationenvironment until a human neural cell or human cell culture enriched inneural cells is produced. In other embodiments, the resultant cells aresubsequently contacted with one or more differentiation medium afterbeing removed from the essentially serum free cell differentiationenvironment until a human neural cell or human cell culture enriched inneural cells is produced, wherein each medium is appropriate to the celltypes as they appear from the preceding cell type. In other embodimentsthe resultant cells are subsequently contacted with a different celldifferentiation environment after being removed from the essentiallyserum free cell differentiation environment until a human neural cell orhuman cell culture enriched in neural cells is produced, wherein eachcell differentiation environment is appropriate to the cell types asthey appear from the preceding cell types.

The invention further provides a composition comprising a culture ofneural cells comprising a neural cell derived in vitro from apluripotent or multipotent cell. In preferred embodiments, the neuralcells are human cells. In preferred embodiments, the neural cell iscapable of expressing one or more of the detectable markers for tyrosinehydroxylase (TH), vesicular monamine transporter (VMAT) dopaminetransporter (DAT), and aromatic amino acid decarboxylase (AADC, alsoknown as dopa decarboxylase). Preferably, the cultured cell expressesdetectable markers for TH, VMAT, DAT, and AADC. In other embodiments,the neural cell is capable of expressing one or more of the detectablemarkers for nestin, Sox1, and Map2. Preferably, the cultured cellexpresses detectable markers for nestin, Sox1, and Map2. Such a cultureof cells not previously provided in the art can be produced by themethods described herein or by other methods later developed.

The present invention further provides a method of culturing a humanpluripotent cell, comprising the steps: a) selecting a human pluripotentcell using an anti-SSEA4 antibody; and b) maintaining a culture of thecell by passaging the cell using a protease treatment, wherein the cellsof the culture express SSEA3, SSEA4, Oct4, Tra-1-60, Tra-1-80, andexpress nestin substantially uniformly. As used herein, the term“substantially uniformly” refers to the expression pattern of a cellularmarker when a colony of cells is examined for expression of that marker.If there is “substantially uniform” expression of a marker, generallymost of the cells of the colony express the marker. For example, if thecenter of an HESC colony does not express a marker, but the marker isexpressed in most of the cells in the remainder of the colony, themarker is not expressed in a substantially uniform manner. Preferably,greater than 90% of the cells of a colony express the marker, morepreferably, greater than 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, of thecells of the colony express the marker, and still more preferably,greater than 99% of the cells of the colony express the marker.

It is to be understood that as used in the specification and in theclaims, “a” or “an” can mean one or more, depending upon the context inwhich it is used. Thus, for example, reference to “a cell” can mean thatat least one cell can be utilized. As also used herein, the term “MEDIIconditioned medium” refers to a medium comprising one or more bioactivecomponents as described herein. In a preferred embodiment, the bioactivecomponent is derived from a hepatic or hepatoma cell or cell lineculture supernatant. The hepatic or hepatoma cell or cell line can befrom any species, however, preferred cell lines are mammalian or avianin origin. The hepatic or hepatoma cell line can be selected from, butis not limited to, the group consisting of: a human hepatocellularcarcinoma cell line such as a Hep G2 cell line (ATCC HB-8065) orHepa-1c1c-7 cells (ATCC CRL-2026); a primary embryonic mouse liver cellline; a primary adult mouse liver cell line; a primary chicken livercell line; and an extraembryonic endodermal cell line such as END-2 andPYS-2. A particularly preferred cell line is the Hep G2 cell line (ATCCHB-8065). A description of the isolation of an essentially serum freeMEDII medium from a Hep G2 cell line is provided in Example 1 below. Inone embodiment of the present invention, the MEDII medium is derivedfrom a Hep G2 cell line and contains supplements of FGF2 and hLIF.

As used herein, the terms “bioactive component” and “bioactive factor”refer to any compound or molecule that induces a pluripotent cell tofollow a differentiation pathway toward an EPL cell or a neural cell.Alternatively, the bioactive component may act as a mitogen or as astabilizing or survival factor for a cell differentiating towards an EPLcell or neural cell. A bioactive component from the conditioned mediummay be used in place of the MEDII conditioned medium in any embodimentdescribed herein. While the bioactive component may be as describedbelow, the term is not limited thereto. The term “bioactive component”as used herein includes within its scope a natural or synthetic moleculeor molecules which exhibit(s) similar biological activity, e.g. amolecule or molecules which compete with molecules within theconditioned medium that bind to a receptor on ES or EPL cells or theirdifferentiation products in adherent culture, in embryoid bodies, or innon-adherent cultures, responsible for EPL or neural induction.

The MEDII conditioned medium described herein can comprise one or morebioactive components selected from the group consisting of a largemolecular weight extracellular matrix protein; a low molecular weightcomponent comprising proline; a biologically active fragment of any ofthe aforementioned proteins or components; and an analog of any of theaforementioned proteins or components. The large molecular weightextracellular matrix protein preferably has a molecular weight ofgreater than approximately, 10 kD, more preferably between approximately100-500 kD and most preferably between approximately 210-250 kD asmeasured on a 10% reducing/denaturing polyacrylamide gel. In a furtherpreferred embodiment, the large molecular weight extracellular matrixprotein comprises a cellular fibronectin protein or a laminin protein.In one preferred embodiment, the bioactive component of the MEDIIconditioned medium can be replaced, at least in part, by proline.Preferably proline is present in the cell culture medium at aconcentration of from approximately 1 μM to approximately 1 M, morepreferably from a concentration of from approximately 5 μM toapproximately 500 μM, more preferably from approximately 10 μM toapproximately 200 μM, and more preferably from approximately 25 μM toapproximately 100 μM. In a preferred embodiment, proline is present inthe cell culture medium at a concentration of approximately 50 μM. Inaddition, the MEDII conditioned medium may contain a neural inducingfactor.

The low molecular weight component of the MEDII conditioned medium cancomprise one or more proline residues or a polypeptide containingproline residues. As used herein, the term “polypeptide” refers to anyof various amides that are derived from two or more amino acids bycombination of the amino group of one acid with the carboxyl group ofanother and usually obtained by partial hydrolysis of proteins. In apreferred embodiment, the low molecular weight component is L-proline ora polypeptide including L-proline. The proline containing polypeptidepreferably has a molecular weight of less than approximately 5 kD, morepreferably less than approximately 3 kD. In a further preferredembodiment, the low molecular weight component is a polypeptide ofbetween approximately 2-11 amino acids, more preferably of betweenapproximately 2-7 amino acids and most preferably approximately 4 aminoacids. The proline containing polypeptide can be selected from, but isnot limited to, the following polypeptides: Pro-Ala, Ala-Pro,Ala-Pro-Gly, Pro-OH-Pro, Pro-Gly, Gly-Pro, Gly-Pro-Ma, Gly-Pro-Ma,Gly-Pro-OH-Pro, Gly-Pro-Arg-Pro (SEQ ID NO:1), Gly-Pro-Gly-Gly (SEQ IDNO:2), Val-Ala-Pro-Gly (SEQ ID NO:3), Arg-Pro-Lys-Pro (SEQ BD NO:4), andArg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-MetOH (SEQ ID NO:5).

In a preferred embodiment, the pluripotent or multipotent cell is ahuman cell. As used herein, the term “pluripotent human cell”encompasses pluripotent cells obtained from human embryos, fetuses oradult tissues. In one preferred embodiment, the pluripotent human cellis a human pluripotent embryonic stem cell. In another embodiment thepluripotent human cell is a human pluripotent fetal stem cell, such as aprimordial germ cell. In another embodiment the pluripotent human cellis a human pluripotent adult stem cell. As used herein, the term“pluripotent” refers to a cell capable of at least developing into oneof ectodermal, endodermal and mesodermal cells. As used herein the term“pluripotent” refers to cells that are totipotent and multipotent. Asused herein, the term “totipotent cell” refers to a cell capable ofdeveloping into all lineages of cells. The term “multipotent” refers toa cell that is not terminally differentiated. The pluripotent human cellcan be selected from the group consisting of a human embryonic stem (ES)cell; a human inner cell mass (ICM)/epiblast cell; a human primitiveectoderm cell, such as an early primitive ectoderm cell (EPL); a humanprimordial germ (EG) cell; and a human teratocarcinoma (EC) cell. Thehuman pluripotent cells of the present invention can be derived usingany method known to those of skill in the art. For example, the humanpluripotent cells can be produced using dedifferentiation and nucleartransfer methods. Additionally, the human ICM/epiblast cell or theprimitive ectoderm cell used in the present invention can be derived invivo or in vitro. EPL cells may be generated in adherent culture or ascell aggregates in suspension culture, as described in WO 99/53021.Furthermore, the human pluripotent cells can be passaged using anymethod known of those to skill in the art, including, manual passagingmethods, and bulk passaging methods such as antibody selection andprotease passaging.

As used herein, the term “neural cell” includes, but is not limited to,a neurectoderm cell; an EPL derived cell; a glial cell; a neural cell ofthe central nervous system such as a dopaminergic cell, an astrocyte oran oligodendrocyte; a neural cell of the peripheral nervous system. Asused herein, the term “neurectoderm” refers to undifferentiated neuralprogenitor cells substantially equivalent to cell populations comprisingthe neural plate and/or neural tube; or a partially differentiatedneural progenitor cell. Neurectoderm cells are multipotent. Therefore,the use of the term “neural cell” in the context of the presentinvention means that the cell is at least more differentiated towards aneural cell type than the pluripotent cell from which it is derived.Also as used herein, producing a neural cell encompasses the productionof a cell culture that is enriched for neural cells. In preferredembodiments, the term “enriched” refers to a cell culture that containsmore than approximately 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or95% of the desired cell lineage. In some embodiments of the presentinvention, neural cells express one or more of the detectable markersTH, VMAT, DAT, and AADC.

The step of contacting the human pluripotent cell or cell culture withthe MEDII conditioned medium to produce embryoid bodies (EBs) or EPLcells can be conducted in any suitable manner. For example, EPL cellsmay be generated in adherent culture or as cell aggregates in suspensionculture. EBs may be generated in suspension culture using the hangingdrop technique or by culturing the cells on agarose coated plates. It isalso to be understood that the step of contacting the embryoid body withan essentially serum free medium and/or an essentially serum free celldifferentiation environment can also be conducted in any manner known tothose of skill in the art. If the embryoid body is contacted with afurther differentiation medium in addition to the essentially serum freedifferentiation medium, it is preferable that the essentially serum freemedium is first removed.

As used herein “essentially serum free” refers to a medium that does notcontain serum or serum replacement, or that contains essentially noserum or serum replacement. As used herein, “essentially” means that ade minimus or reduced amount of a component, such as serum, may bepresent that does not eliminate the improved bioactive neural cellculturing capacity of the medium or environment. For example,essentially serum free medium or environment can contain less thanapproximately 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% serum whereinthe presently improved bioactive neural cell culturing capacity of themedium or environment is still observed. In preferred embodiments of thepresent invention, the essentially serum free medium does not containserum or serum replacement.

In one preferred embodiment, the essentially serum free differentiationmedium comprises MEDII conditioned medium. Preferably, the essentiallyserum free differentiation medium comprises approximately 20% toapproximately 80% MEDII conditioned medium, more preferably theessentially serum free differentiation medium comprises approximately30% to approximately 70% MEDII conditioned medium, still more preferablythe essentially serum free differentiation medium comprisesapproximately 40% to approximately 60% MEDII conditioned medium, andmost preferably, the essentially serum free differentiation mediumcomprises approximately 50% MEDII conditioned medium. In otherembodiments, the essentially serum free differentiation medium comprisesapproximately 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or80% MEDII conditioned medium.

As used herein “essentially LIF free” refers to a medium that does notcontain leukemia inhibitory factor (LIF), or that contains essentiallyno LIF. “Essentially’ means that a de minimus or reduced amount of acomponent, such as LIF, may be present that does not eliminate theimproved bioactive neural cell culturing capacity of the medium orenvironment. For example, essentially LIF free medium or environment cancontain less than approximately 100, 75, 50, 40, 30, 10, 5, 4, 3, 2, or1 ng/ml LIF, wherein the presently improved bioactive neural cellculturing capacity of the medium or environment is still observed. Asused herein, the term “LIF” refers to leukemia inhibitory factor that isobtained or derived from any species, and is therefore not limited tohuman LIF.

In certain embodiments of the above methods, the MEDII conditionedmedium is essentially free from LIF and from FGF2. In other embodimentsof the above methods, the first or subsequent differentiation media areessentially free from LIF and from FGF2.

In one embodiment of the present inventions, the pluripotent cell orcell culture is cultured with a minimal medium. As used herein, the term“minimal medium” refers to a tissue culture medium that is preferablyessentially free from FGF, proline, and/or MEDII. As used herein,“essentially free from FGF” or “essentially FGF free” refers to a tissueculture medium that contains less than approximately 10, 9, 8, 7, 6, 5,4, 3, 2, 1, 0.5, 0.1, or 0.01 ng/ml of an FGF, wherein the presentlyimproved bioactive neural cell culturing capacity of the medium orenvironment is still observed. Preferably, the minimal medium comprisesless than 1 ng/ml of an FGF. As used herein, “essentially free fromproline” or “essentially proline free” refers to a tissue culture mediumthat contains less than approximately 500 μM, 400 μM, 300 μM, 200 μM,100 μM, 50 μM, 10 μM, 5 μM, or 1 μM of proline. In one embodiment, theminimal medium comprises less than 10 μM proline. In another embodiment,the minimal medium is supplemented with proline. When the minimal mediumis supplemented with proline, preferably the proline is present at aconcentration of less than 500 μM, 400 μM, 300 μM, 200 μM, 100 μM, 50μM, 10 μM, 5 μM, or 1 μM of proline. In one embodiment, the minimalmedium comprises approximately 50 μM proline. As used herein,“essentially free from MEDII” or “essentially MEDII free” refers to atissue culture medium that contains less than approximately 50%, 40%,30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% of MEDII, as defined herein.Preferably, a tissue culture medium essentially free from MEDIIcomprises less than 5% MEDII.

As used herein, an “essentially single cell culture” is a cell culturewherein during passaging, the cells desired to be grown are dissociatedfrom one another, such that the majority of the cells are single cells,or two cells that remain associated (doublets). Preferably, greater than50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of the cellsdesired to be cultured are singlets or doublets.

In a preferred embodiment of the above methods, a “feeder cell” is acell that is co-cultured with a human pluripotent cell and maintains thehuman pluripotent cell in an undifferentiated or partiallydifferentiated state. In a preferred embodiment of the above method, theconditioned medium is obtained from a feeder cell that maintains thehuman pluripotent cell in an undifferentiated or partiallydifferentiated state. In one embodiment, the feeder cell is a mousecell, such as a mouse embryonic fibroblast. In a preferred embodiment,the mouse embryonic fibroblast is mitotically inactivated, using methodswell known to those of skill in the art. In another embodiment, thefeeder cell is a human feeder cell. In certain embodiments, the humanfeeder cell is selected from the group consisting of a human fibroblastcell, a MRC-5 cell, a human embryonic kidney cell, a mesenchymal cell,an osteosarcoma cell, a keratinocyte, a chondrocyte, a Fallopian ductalepithelial cell, a liver cell, a cardiac cell, a bone marrow stromalcell, a granulosa cell, a skeletal muscle cell, and an aorticendothelial cell. In a more preferred embodiment the human feeder cellis selected from the group consisting of a skin keloid fibroblast cell,a fetal skin fibroblast cell, a bone marrow stromal cell, or a skeletalmuscle cell.

The present invention contemplates that the feeder cell is a freshlyplated feeder cell. As used herein, the term “freshly plated” means thatthe feeder cell has been allowed to attach to the tissue culture dishfor less than 2 days. Preferably, the feeder cell has been plated forless than 18 hours, more preferably the feeder cell has been plated forless than 10 hours, more preferably the feeder cell has been plated forless than 6 hours, and most preferably, the feeder cell has been platedfor less than 2 hours. In another embodiment, preferably the feeder cellhas been plated for approximately 6 to 18 hours. In a preferredembodiment, HESC cultures that have been protease passaged and/orantibody selected are prepared for differentiation by seeding the cellsat a defined density on feeder layers that are between approximately 6to 18 hours old. In another embodiment, manually passaged HESC culturesare prepared for differentiation by seeding the cells at a defineddensity on feeder layers that are freshly plated. Seeding manuallypassaged HESCs on fresh feeder layers appears to cause a differentiationevent that enables uniform neural rosette differentiation in suspension,and although morphological changes are not apparent, may also have apositive influence on the neural and DA differentiation of bulk passagedHESC.

As stated above, the present invention provides a method of producing aneural cell or enriching a culture for neural cells comprising the stepsof: 1) providing a pluripotent human cell; 2) culturing the pluripotenthuman cell with an essentially serum free medium comprising MEDIIconditioned medium and forming an embryoid body; and 3) culturing theembryoid body with an essentially serum free cell differentiationenvironment, and optionally contacting the embryoid body with one ormore subsequent cell differentiation environments, until the neural cellor cell culture enriched in neural cells is produced.

It is to be understood that the step of contacting the pluripotent cellwith the MEDII conditioned medium includes the use of a “normal” or“other” medium supplemented with a MEDII conditioned medium. The“normal” or “other” medium, such as a normal primate ES medium, can besupplemented with a MEDII conditioned medium at any concentration, butit is preferred that the “normal” or “other” medium is supplemented atbetween approximately 10-75%, more preferably between approximately40-60% and most preferably approximately 50% MEDII conditioned medium.In one embodiment, the pluripotent human cell is in contact with theMEDII conditioned medium between approximately 1-60 days, morepreferably between approximately 2-15 days, and most preferably 5-10days. In a further preferred embodiment, the pluripotent cells are notpassaged during the MEDII incubation.

Following MEDII incubation, cells from the embryoid body can becontacted with an essentially serum free cell differentiation medium asdescribed above. The cells from the embryoid body can be in contact withthe essentially serum free cell differentiation medium for approximatelyone or more days, but preferably approximately three or more days. Whenthe cells from the embryoid body are subsequently contacted with asubsequent differentiation medium as described above, such contact canoccur for approximately one or more days, but preferably approximatelythree or more days. The human neural cells generated using thecompositions and methods of the present invention can be generated inadherent culture or as cell aggregates in suspension culture. It is tobe understood that the methods of the present invention can comprise thesequential use of adherent cultures and suspension culture. Preferably,the human neural cells are produced in suspension culture.

As used herein, the term “cell differentiation environment” refers to acell culture condition wherein the pluripotent cells or embryoid bodiesderived therefrom are induced to differentiate into neural cells, or areinduced to become a human cell culture enriched in neural cells.Preferably the cell lineage induced by the cell differentiationenvironment will be homogeneous in nature. As used herein, the term“homogeneous,” refers to a population that contains more thanapproximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% of the desired neuronal cell lineage.

In one embodiment, the cell differentiation environment is a suspensionculture where the medium is Dulbecco's Modified Eagle's Mediums andHam's F12 media (DMEM/F12), and comprises a fibroblast growth factor(FGF) such as FGF-2. In a preferred embodiment the cell differentiationenvironment is a suspension culture where the medium is DMEM/F12, FGF-2,and MEDII conditioned media. In a preferred embodiment, the suspensionculture is an agarose suspension culture. In one embodiment, the celldifferentiation environment is also essentially free of human leukemiainhibitory factor (hLIF). In certain preferred embodiments, the celldifferentiation environment is essentially serum free.

The essentially serum free cell differentiation environment can alsocontain supplements such as L-Glutamine, NEAA (non-essential aminoacids), P/S (penicillin/streptomycin), N2 and β-mercaptoethanol (β-ME).It is contemplated that, additional factors may be added to the celldifferentiation environment, including, but not limited to fibronectin,laminin, heparin, heparin sulfate, retinoic acid, members of theepidermal growth factor family (EGFs), members of the fibroblast growthfactor family (FGFs) including FGF2 and/or FGF8, members of the plateletderived growth factor family (PDGFs), transforming growth factor(TGF)/bone morphogenetic protein (BMP)/growth and differentiation factor(GDF) factor family antagonists including but not limited to noggin,follistatin, chordin, gremlin, cerberus/DAN family proteins, ventropin,and amnionless. TGF/BMP/GDF antagonists could also be added in the formof TGF/BMP/GDF receptor-Fc chimeras. Other factors that may be addedinclude molecules that can activate or inactivate signaling throughNotch receptor family, including but not limited to proteins of theDelta-like and Jagged families as well as gamma secretase inhibitors andother inhibitors of Notch processing or cleavage. Other growth factorsmay include members of the insulin like growth factor family (IGF), thewingless related (WNT) factor family, and the hedgehog factor family.Additional factors may be added to promote neural stem/progenitorproliferation and survival as well as neuron survival anddifferentiation. These neurotrophic factors include but are not limitedto nerve growth factor (NGF), brain derived neurotrophic factor (BDNF),neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5), interleukin-6 (IL-6),ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF),cardiotrophin, members of the transforming growth factor (TGF)/bonemorphogenetic protein (BMP)/growth and differentiation factor (GDF)family, the glial derived neurotrophic factor (GDNF) family includingbut not limited to neurturin, neublastin/artemin, and persephin andfactors related to and including hepatocyte growth factor.

In other embodiments, the cell differentiation environment comprisesseeding the embryoid body to an adherent culture. As used herein, theterms “seeded” and “seeding” refer to any process that allows anembryoid body or a portion of an embryoid body to be grown in adherentculture. An used herein, the term “a portion” refers to at least onecell from an embryoid body, preferably between approximately 1-10 cells,more preferably between approximately 10-100 cells from an embryoidbody, and more preferably still between approximately 50-1000 cells froman embryoid body. As used herein, the term “adherent culture” refers toa cell culture system whereby cells are cultured on a solid surface,which may in turn be coated with a substrate. The cells may or may nottightly adhere to the solid surface or to the substrate. The substratefor the adherent culture may further comprise any one or combination ofpolyornithine, laminin, poly-lysine, purified collagen, gelatin,extracellular matrix, fibronectin, tenascin, vitronectin, polyglycolytic acid (PGA), poly lactic acid (PLA), poly lactic-glycolic acid(PLGA) and feeder cell layers such as, but not limited to, primaryastrocytes, astrocyte cell lines, glial cell lines, bone marrow stromalcells, primary fibroblasts or fibroblast cells lines. In addition,primary astrocyte/glial cells or cell lines derived from particularregions of the developing or adult brain or spinal cord including butnot limited to olfactory bulb, neocortex, hippocampus, basaltelencephalon/striatum, midbrain/mesencephalon, substantia nigra,cerebellum or hindbrain may be used to enhance the development ofspecific neural cell sub-lineages and neural phenotypes. Furthermore,the substrate for the adherent culture may comprise the extracellularmatrix laid down by a feeder cell layer, or laid down by the pluripotenthuman cell or cell culture.

The human neural cells produced using the methods of the presentinvention have a variety of uses. In particular, the neural cells can beused as a source of nuclear material for nuclear transfer techniques,and used to produce cells, tissues or components of organs fortransplant. The neural cells of the present invention can also be usedin human cell therapy or human gene therapy to treat neuronal diseasessuch as Parkinson's disease, Huntington's disease, lysosomal storagediseases, multiple sclerosis, memory and behavioral disorders,Alzheimer's disease and macular degeneration. Other pathologicalconditions including stroke and spinal cord injury can be treated usingthe neural cells of the present invention. The neural cells can also beused in testing the effect of molecules on neural differentiation orsurvival, in toxicity testing or in testing molecules for their effectson neural or neuronal functions. This could include screens to identifyfactors with specific properties affecting neural or neuronaldifferentiation, development, survival or function. In this applicationthe cell cultures could have great utility in the discovery, developmentand testing of new drugs and compounds that interact with and affect thebiology of neural stem cells, neural progenitors or differentiatedneural or neuronal cell types.

The neural cell or the human cell culture enriched in neural cells maydisperse and differentiate in vivo following brain implantation. Inparticular, following intraventricular implantation, the cell can becapable of dispersing widely along the ventricle walls and moving to thesub-ependymal layer. The cell can be further able to move into deeperregions of the brain, including into the untreated (e.g., by injection)side of the brain into sites that include the thalamus, frontal cortex,caudate putamen and colliculus. In addition the neural cell or humancell culture enriched in neural cells can be injected directly intoneural tissue with subsequent dispersal of the cells from the site ofinjection. This could include any region, nucleus, plexus, ganglion orstructure of the central or peripheral nervous systems.

The invention further provides methods of producing a partiallydifferentiated pluripotent cell comprising culturing pluripotent cellson a layer of fresh feeder cells, wherein the fresh feeder cells areless than 2 days old, thereby inducing formation of a moredifferentiated pluripotent cell. In preferred embodiments, the freshfeeder cells are less than one day old, more preferably less than 12hours old, or more preferably less than 6 hours old. In preferredembodiments, the more differentiated pluripotent cell is obtained fromthe central, or crater, region of the colony of pluripotent cells. Insome embodiments, the more differentiated pluripotent cell expressesless Oct4 marker than an embryonic stem cell. The invention furtherprovides a composition comprising these more differentiated pluripotentcells.

Throughout this application, various publications are referenced. Thedisclosures of all of these publications and those references citedwithin those publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art to which this invention pertains. In particular, U.S.Provisional Patent Application Nos. 60/401,968 and 60/459,090, andInternational Application PCT/AU03/00552 are hereby incorporated byreference in their entirety. The following examples are not intended tolimit the scope of the claims to the invention, but are rather intendedto be exemplary of certain embodiments.

EXAMPLES Example 1

Production of Essentially Serum Free MEDII Conditioned Media

An essentially serum free MEDII conditioned medium was produced asfollows. Hep G2 cells (Knowles et al., 1980 Nature 288:615-618; ATCCHB-8065) were seeded at a density of 5×104 cells/cm2 and cultured forthree days in DMEM. Cells were washed twice with 1×PBS and once withserum free medium (DMEM containing high glucose but without phenol red,supplemented with 1 mM L-glutamine, 0.1 mM β-ME, 1×ITSS supplement(Boehringer Mannheim), 10 mM HEPES, pH 7.4 and 110 mg/L sodium pyruvate)for 2 hours. Fresh serum free medium was added at a ratio of 0.23 ml/cm2and the cells were cultured for a further 3-4 days. SfMEDII wascollected, sterilized and stored. A further explanation of MEDIIconditioned media can be found in International Application No. WO99/53021, herein incorporated by reference in its entirety.

Example 2

Isolation of the Neural Inducing Component of MEDII Media

Essentially serum free MEDII (sfMEDII) or serum containing MEDII is usedas a source of the biologically active factor in all purificationprotocols in this examples. The bioactive component of MEDII, referredto as the neural inducing factor, is routinely isolated from the sfMEDIIor MEDII using purification techniques well known in the art. Thesetechniques can include ultrafiltration; column chromatography, includingion exchange columns, hydrophobic columns, hydroxyapatite andgel-filtration columns; affinity chromatography; high performance liquidchromatography (HPLC); or FPLC. After each step of the purificationprotocol, individual samples are assayed directly for the biologicalactivity of the neural inducing factor on ES cells. Reducing SDS PAGEcan reveal the presence of a highly purified component in samplescontaining the neural inducing factor bioactivity.

The purified and isolated neural inducing component is capable ofindefinite storage, and is used in place of MEDII conditioned medium toinduce the formation of neural cells from pluripotent or multipotentstem cells, or to enrich a human cell culture for neural cells.

Example 3

Derivation and Characterization of a Neurosphere Population from MouseEmbryonic Stem Cells

Preparation of Embryoid Bodies from Mouse ES Cells

D3 mouse embryonic stem cells were maintained on gelatin free tissueculture plates and passaged every three to four days. The mouse ES cellculture medium was 10% fetal bovine serum (FBS), Dulbecco's ModifiedEagles Medium (DMEM), 0.1M β-mercaptoethanol (β-ME), 1 mM glutamine,1000 U/ml mouse LIF (ESGRO). The ES cell colonies were rinsed twice withPBS and treated with Trypsin/EDTA for one minute, they were thentriturated and blocked with an equal volume of FBS, and thencentrifuged, resuspended and counted.

Embryoid bodies were formed by seeding the ES single cell suspension at1×10⁵ cells/ml in IC:DMEM media (10% FBS, 90% DMEM, 1 mM Glutamine, and0.1% β-ME). Embryoid bodies were allowed to aggregate for two days, andwere then split 1:2 in IC:DMEM media (EB²), cultured for a further 2days and split again 1:2 (EB⁴) and then cultured for three further dayswith daily changes of media (EB⁷). The culture conditions were thenchanged to essentially Serum-Free media (50% DMEM, 50% Ham's F12 (GibcoBRL), 1×ITSS (Boehringer-Mannheim) and 10 ng/ml FGF-2 (Peprotech Inc.)for a further eight days (EB¹⁵).

To produce MEDII treated embryoid bodies (EBMs), a single cellsuspension of ES cells was seeded at 1×10⁵ cells/ml in IC:DMEMsupplemented with MEDII (50% MEDII). The MEDII conditioned medium usedin the experiments described in this Example was produced in the samemanner as the essentially serum free MEDII conditioned medium describedin Example 1, except that it contained 5% fetal calf serum. EBMs wereallowed to aggregate for two days and then were split 1:2 in 50% MEDIImedia (EBM²), cultured for a further 2 days and split 1:2 (EBM⁴, EPLcells in suspension and then cultured for three further days with dailychanges of 50% MEDII media (EBM⁷). After day seven, the medium was thenchanged to Serum-Free medium as it was for EBs for a further eight days(EBM¹⁵).

All-Trans Retinoic Acid Supplementation

For treatments involving all-trans retinoic acid (RA), a 4−/4+ culturesupplementation method was used. Briefly, both EB and EBM cellaggregates were allowed to form in appropriate media for four days, andthe media was then supplemented with 100 nM all-trans retinoic acid(Sigma) with each daily media change thereafter for a further four days.

Generation of Neurosphere Suspension Cultures

Both EBs and EBMs were triturated to a near single cell suspension afterbeing cultured for periods of 7, 9, 12, and 15 days (7 days in MEDIIfollowed by an appropriate number of days in essentially Serum-Freemedia). Two methods of trituration, either mechanical dissociation ortrypsin dissociation, yielded similar results. Cells were seeded atapproximately 10-20 cells/μl of media into 10 mls of neurosphere media(DMEM:F12, 10 ng/ml FGF-2, 10 μg/ml heparin (SIGMA), 1/50 B27 (GibcoBRL), 1/100 penicillin/streptomycin, 1/100 ITSS) in a T75 culture flask.Cultures were maintained in this media with a 50:50 change of freshmedia after 7 days and cultured for 10-12 days. Sphere formation wasreadily apparent after three days in culture, and robust spheres hadformed by day 7. One population of spheres derived from EBM¹² (no RA)aggregates was passaged and grown for a further two passages to yieldtertiary spheres.

The tertiary spheres were seeded onto Poly-L-Ornithine/Fibronectincoated chambers slides (Nunc) and cultured in neurosphere medium forthree days before fixing and processing for three representativeneuronal lineage immunohistochemical markers, NF200, O4, and GFAP.

Grafts into the Rat Striatum

To prevent graft rejection of implanted mouse cells rats received dailyimmunosuppression by i.p. injection of cyclosporin A diluted in extravirgin olive oil (1 mg/kg Sandimmune, Sandoz Pharmaceutical). Dailyinjections started one day prior to the implantation of cells. Rats wereintubated and anesthesia was maintained (Ventilator 74 strokes/min, 2 mlstrokes, halothane 1-1.5%, oxygen flow rate at less than 1 liter/min).Animals were mounted in a Kopf Stereotaxic frame (Kopf Instruments,Tujanga, Calif.), the head was shaved and Betadine applied to the shavenarea. An incision was made to expose bregma and lambda and the nose baradjusted to level bregma and lambda. Each injected animal received a 1μl volume (1000-2000 cells/μl) delivered over 1 minute into the rightstriatum using a 5 μl SGE positive displacement syringe and a 23 Gneedle (SGE International, Victoria, Australia). Striatal coordinateswere: anterior 0.9 mm, lateral 2.7 mm, depth 5.0 mm. The needle was leftin place for 4 minutes and removed over a one minute period. The rat wassutured and Xylocaine and Ilium Dermapred were applied to the sutureline. The intubation tube was removed and the rat was allowed to recoveron a heating pad. A subcutaneous injection of butorphanol (2 ml/kg) wasadministered as a post-operative analgesic. Engrafted rats were left fora period of 4 weeks and brains were harvested by transcardial perfusionwith PBS and 4% PFA, dissecting away the skull. The brains were storedin PBS containing 0.01% sodium azide.

Results

Formation of neurospheres differed dramatically between single cellsuspensions generated from either ES cell aggregates grown in eitherIC:DMEM (EBs) or grown in 50% MEDII (EBMs). From Table 1, it is shownthat ES cell aggregates formed in IC:DMEM followed by periods of serumstarvation exhibit poor sphere forming capacity even when treated withthe potent neural inducer, all-trans retinoic acid. In contrast, a MEDIIdependent effect was observed in cell aggregates that had formed in 50%MEDII followed by a period of serum starvation. Robust sphere formingcapacity was clearly seen in EBM¹² aggregates with sphere formationvisible after 34 days in neurosphere culture media. The capacity forsphere formation seemed to be diminished in EBMs on either side of thistime frame. An effect of all-trans retinoic acid was observed such thatsphere forming capacity seemed to emerge earlier at EBM⁹, and show adecrease with further culturing. In both cases, robust sphere formingcapacity was only seen in cells derived from embryoid bodies that hadbeen conditioned in 50% MEDII. TABLE 1 Neurosphere formation isdependent on MEDII, and is influenced by retinoic acid. Day 9 Day 12 Day15 (50% MEDII or (50% MEDII or (50% MEDII or Day 7 IC:DMEM plus IC:DMEMplus IC:DMEM plus (50% MEDII or 2 days essentially 5 days essentially 8days essentially IC:DMEM only) serum free) serum free) serum free) EB −− − − EB + RA − − − − EBM + + +++ ++ EBM + RA + +++ + +EB = embryoid body;EBM = embryoid body cultures in 50% MEDII;RA = 100 nM all-trans retinoic acid.“−“ indicates no or very poor sphere formation,“+” indicates poor sphere forming capacity,“++” indicates moderate sphere forming capacity, and“+++” indicates high sphere forming capacity.

Sphere formation was observed after two further passages of EBM¹² (noRA) spheres that were mechanically passaged and reseeded at a 10-20cells/μl density in; neurosphere media. During the passaging of thesecells it was noted that dense networks of cells formed on the bottom ofthe flask where sphere had attached. These networks exhibited extensiveneural morphology and extensive networks of neurites were observed.Dense clusters of cells appeared and were likely to act as a source ofmore spheres. Spheres from these tertiary passaged spheres were seededonto glass chamber slides and allowed to grow for three days beforeprocessing for immunohistochemistry. These single spheres grew to formsimilar extensive networks of cells with dense regions that appeared tobe forming more spheres. Marker analysis revealed that there were largenumbers of GFAP positive astrocytic lineage cells, moderate numbers ofNF200 positive neurons, and low levels of 04 positive oligodendrocytes.The seeded spheres were therefore capable of producing cells of allthree neural lineages after three passages at clonal cell densities andtherefore provide evidence of self-renewal and multi-potency.

An in vivo analysis of low cell number grafts into the rat Striatumrevealed detectable mouse cells that line the needle tract of theinjection site. No obvious signs of gross teratoma formation werevisible and the number of detectable cells was low (10-20 per 10 μMsection through the graft site).

Example 4

Derivation and Characterization of a Neurosphere Population from MouseEmbryonic Stem Cells Using a Two-Stage Process

Example 3 was generally repeated, however, a two-stage process wasfollowed with cells grown as aggregates/embryoid bodies in the one media(Stage A) prior to disaggregation for Stage B growth conditions. MouseES cells were separated by treatment with trypsin and the single ES cellsuspension seeded in a cellular aggregate culture media (DMEM:F12 andeither N2 or ITSS) that was free of serum. The addition of 10 ng/ml FGF2and/or 100 nM dose of RA is optional. Alternatively the cellaggregates/embryoid bodies are formed in the presence of DMEM and 100 μMproline with either N2 or ITSS and optionally with FGF2 and RA. Cellaggregates/embryoid bodies were allowed to form in Costar low attachmenttissue culture dishes for a period of up to 15 days in suspensionculture (Stage A) and then were triturated to a single cell suspensionusing trypsin dissociation.

Cells were then seeded at a concentration of less than 100 cells/μL inneurosphere media (DMEM:F12, 10 ng/ml FGF2, 10 μg/ml heparin (SIGMA),1/50 B27 (GIBCO), 1/100 pen/strep, 1/100 ITSS) in a T75 culture flask.Cultures were maintained in this media for 14 to 21 days (Stage B) andthe neurospheres so formed can be maintained by passaging in the samemedia.

These cell aggregates tended to demonstrate an irregular appearance ofthe cell layer. Robust embryoid body formation with morphologicalcharacteristics of neurectoderm did not occur without the presence ofF12 media.

The neurospheres formed can be seeded onto poly-L-ornithine/laminincoated plates and allowed to adhere and differentiate. Optionally inthis culture stage, neurospheres are maintained in media containingcombinations of RA, 50% MEDII and L-proline. In an alternativetreatment, during stage A, the embryoid bodies (EB9) are seeded ontopoly-L-ornithine/laminin coated culture plates and cultured for 6 to 8days to permit neuronal differentiation.

In an alternative treatment, embryoid bodies cultured from stage A aretriturated and resuspended in a minimal media (DMEM/F12 and N2 or ITSS).Optionally, this media includes combinations of FGF, MEDII, RA andL-proline. The aggregates formed can also be seeded ontopoly-L-ornithine/laminin coated plates and allowed to adhere anddifferentiate.

Example 5

Formation and Characterization of EBs from Human ES Cells in EssentiallySerum Free Conditions

Manual Passaging of Human ES Cells

Human embryonic stem cells (HESCs) identified as BGN01 and BGN02(BresaGen, Inc. Athens, Ga.) were used in this work. The HESCs weregrown in DMEM/F12 (50/50) supplemented with 15% FBS, 5% knockout serumreplacer (Invitrogen), 1× non-essential amino acids (Invitrogen),L-Glutamine (20 mM), penicillin (0.5 U/ml), streptomycin (0.5 U/ml),human LIF (10 ng/ml, Chemicon) and FGF-2 (4 ng/ml, Sigma). The human EScells were grown on feeder layers of mouse primary embryonic fibroblaststhat were mitotically inactivated by treatment with mitomycin-C. Feedercells were re-plated at 1.2×10⁶ cells per 35 mm dish. The mitoticallyinactivated fibroblasts were cultured for at least 2 days prior to theplating of HESCs. The HESCS were manually passaged onto fresh fibroblastfeeder layers every 3-4 days using a fire-pulled Pasteur pipette.Briefly, the barrel of the Pasteur pipette was melted solid and drawnout to a solid needle approximately 1 cm long and approximately 25 μm indiameter, which was sequentially pressed through HESC colonies to form auniform grid of cuts. The same needle was passed under the colonies tolift them from the feeder layer. Entire plates of HESCs were harvested,then the colonies were broken into individual pieces defined by the gridby gentle pipetting using a 5 ml serological pipette. The pieces from asingle plate were split between 2 or 3 new plates that were coated withfeeder layers of mitotically inactivated mouse primary embryonicfibroblasts.

SSEA4 Selection and Bulk Passaging of HESCs

SSEA4 staining appears to be closely associated with theundifferentiated state of HESCs. Undifferentiated domed HESC coloniesshow a uniform distribution of SSEA4 immunostaining, whiledifferentiating HESC colonies show reduced or no expression of SSEA4 inmorphologically differentiated cells. An example of this is the reducedSSEA4 expression in morphologically differentiated cells that occurswithin the crater cells located in the center of manually passaged HESCsthat are plated onto fresh feeder layers These crater cells grow as amonolayer, surrounded by multilayered morphologically undifferentiatedHESCs. Since SSEA4 appears to be selective for a population ofundifferentiated HESCs, it was chosen to use as a selectable marker.

Undifferentiated HESCs were selected by magnetic sorting using ananti-SSEA4 antibody (Developmental Studies Hybridoma Bank) and the MACSseparation system (Miltenyi Biotec) according to the manufacturersinstructions. Briefly, manually passaged HESCs were harvested bytreating with 1 mg/ml Collagenase (Gibco) for 5 minutes, followed bytreating with 0.05% Trypsin/EDTA for 30 seconds. Colonies were thenflushed off the top of the feeder layer and dissociated to anessentially single cell suspension, leaving the feeder cells behind as anet. The trypsin was neutralized with 10% FBS/10% KSR human ES mediumand passed through a cell strainer (Becton Dickinson). For blocking,cells were pelleted and resuspended in staining buffer (5% FBS, 1 mMEDTA, penicillin (0.5 U/ml) and streptomycin (0.5 U/ml), in Ca2⁺/Mg2⁺free PBS).

The cells were pelleted and resuspended in 1 ml primary anti-SSEA4antibody diluted 1:10 in staining buffer, and incubated at 4° C. for 15minutes. 9 ml staining buffer was then added and the cells werepelleted, washed with 10 ml staining buffer and re-pelleted. 1×10⁷ cellswere resuspended in 80 μl staining buffer and 20 μl magnetic goatanti-mouse IgG MicroBeads were added, mixed and incubated at 4° C. for10 minutes. The volume was then brought to 2 ml with staining buffer and2 μl of a fluorescent conjugated secondary antibody (Alexa-488conjugated goat anti-mouse IgG, Molecular Probes) was added to enablefluorescent analysis of the separation. The sample was incubated for 5minutes at 4° C., then the volume was brought to 10 ml with stainingbuffer and the cells were pelleted and washed in 10 ml staining bufferand re-pelleted. The cells were resuspended in 500 μl staining bufferand applied to a separation column that had been prepared by washing itthree times with 500 μl staining buffer. The column was positioned onthe selection magnet prior to application of the cells and theflow-through and three washes with 500 μl staining buffer werecollected. These cells in these fractions were presumably a SSEA4negative population. The column was removed from the magnet, 500 μlstaining buffer was added and forced through with a plunger, and thepresumed SSEA4 positive cell population was collected in a 15 ml tube.20% KSR human ES growth medium was added to bring the volume to 10 ml,and the cells were pelleted and resuspended in 1 ml of the same medium.10⁵ SSEA4 selected HESCs were plated on 35 mm dishes plated with a mouseembryonic fibroblast feeder layer, and the cells were maintained andpassaged in 20% KSR growth medium (see below).

To examine the effectiveness of the selection, aliquots of the flow/washsample and SSEA4 selected sample were analyzed by fluorescencemicroscopy. Approximately 75% of the cells from the retained fractionwere SSEA4 positive, indicating effective enrichment.

Bulk passaged HESCs were grown in DMEM/F12 (50/50) supplemented with 20%knockout serum replacer (KSR; Invitrogen), 1×NEAA (Invitrogen),L-Glutamine (20 mM), penicillin (0.5 U/ml), streptomycin (0.5 U/ml),human LIF (10 ng/ml, Chemicon) and FGF-2 (4 ng/ml, Sigma). Forpassaging, cells were treated with 1 mg/ml Collagenase (Gibco) for 5minutes, followed by 0.05% Trypsin for 30 seconds and the cells werethen dissociated with a 1 ml pipette. The feeder layer remained as amesh and was removed with a pipette. DMEM/F12 (50/50) supplemented with10% FBS and 10% KSR was added to the HESC suspension, followed bycentrifugation, aspiration and resuspension in culture medium. HESCswere replated at 1×10⁵ cells per 35 mm dish on a feeder layer.

Formation of Essentially Serum Free Embryoid Bodies

HESC cultures were washed once with DMEM/F12 and once with DMEM/F12supplemented with 1×N2 supplement (Invitrogen). Undifferentiated HESCcolonies were harvested by the manual passaging methods described aboveinto uniform colony pieces of approximately 10-100 cells. Pieces weretransferred to 15 ml tubes and washed in 10 ml DMEM/F12 plus 1×N2supplement. The pieces were left to settle, and the medium wasaspirated. The pieces were resuspended in 2.5 ml of medium, andtransferred to suspension dishes.

Suspension dishes were prepared by coating the surface of non-tissueculture plastic Petri dishes with a layer of agarose. The agarosecoating was generated by pouring a molten solution of 0.5% agarose inDMEM/F12 medium into the Petri plates. The agarose coating wasequilibrated in DMEM/F12 medium. Suspension cultures contained 2.5 ml ofmedium for 35 mm dishes, or 10 ml of medium for 100 mm dishes.

Essentially serum free embryoid bodies were cultured in suspension forup to four weeks, with replenishment of the medium every 3-4 days. Theessentially serum free embryoid bodies were passaged every 5-7 days bycutting them into pieces with drawn out solid glass needles. Atpassaging, the embryoid bodies contained approximately 5000-10,000 cellsand were divided into 4-10 pieces. Essentially serum free embryoidbodies formed in the presence of DMEM/F12 with 1×N2 and FGF-2 are termedsfEBs, while essentially serum free embryoid bodies formed in thepresence of DMEM/F12 with 1×N2 and FGF-2 and 50% MEDII are termedsfEBMs.

Clonal Derivation of Colonies from Essentially Serum Free EmbryoidBodies Treated with MEDII Medium

sfEBMs were transferred to tissue culture dishes (4-well or 35 mm) andwere rinsed with Ca²⁺/Mg²⁺ free PBS. After settling, the PBS wasaspirated and 1 or 2 ml of 0.25% trypsin was added. sfEBMs weredispersed to a single cell suspension, with occasional doublets ortriplets, by gentle trituration using a 1 ml pipette. The cellsuspension was transferred to a 15 ml tube, diluted with 9 or 18 ml ofmedium and pelleted by centrifugation. The pellet was resuspended inDMEM/F12 supplemented with 1×N2 supplement, FGF-2 (4 ng/ml), 50% MEDIIand added to agarose coated suspension dishes at low density (0.5-5cells/μl). These cultures were not disturbed for 4-5 days, when smalluniform colonies could be observed. Colonies could be harvested atapproximately 10 days.

Immunohistochemistry

Essentially serum free embryoid bodies (sfEBs and sfEBMs) were cut intopieces using glass needles and 1-15 pieces were plated ontopolyornithine/laminin coated permanox chamber slides in the same mediumused for suspension culture. Polyornithine/laminin coated slides wereprepared by diluting polyornithine to 20 μg/ml in tissue culture gradewater, coating chamber wells at 37° C. overnight, washing twice withwater and coating the chamber wells with 1 μg/ml laminin at 37° C.overnight. The slides were washed with water and 1×PBS prior to platingthe cells. The embryoid bodies were cultured on these slides for 2-7days.

For preparing cytospins, embryoid bodies in suspension weredisaggregated and attached to a glass slide using a standard cytospinapproach for immunostaining (Watson P. A., J. Lab. Clin. Med.68:494-501, 1966). sfEBMs were washed with 1×PBS and disaggregated with0.05% trypsin and gentle trituration. The cell suspension was washedwith culture medium, pelleted and resuspended in HESC medium and 1×104cells were attached to a glass microscope slide by centrifugation at 300g for 4 minutes using a cytospin apparatus (Heraeus Instruments GmbH).The attached cells were fixed immediately with 4% paraformaldehyde, and4% sucrose in 1×PBS for 15 minutes, followed by three separate 5-minutewashes in 1×PBS.

For immunostaining, on fixed cells or cytospins, the samples were rinsedwith 1×PBS and fixed in 4% paraformaldehyde, 4% sucrose in 1×PBS for 30minutes at 4° C. The cells were then washed in 1×PBS and stored at 4° C.To perform immunostaining, the cells were washed in block buffer (3%goat serum, 1% polyvinyl Pyrolidone, 0.3% Triton X-100 in wash buffer)for 30 minutes, and then incubated with the appropriate dilution of theprimary antibody, or combination of antibodies for 4-6 hours at roomtemperature. The primary antibodies were anti-Map2, a mouse monoclonalantibody recognizing the Map-2 a, b and c isoforms (Sigma, Catalog #M4403) at a 1/500 dilution; anti-Sox1, a chicken monospecific polyclonalantibody (Chemicon, Catalog # AB5934) at a 1/250 dilution; andanti-Nestin, a rabbit polyclonal antibody (Chemicon, Catalog # AB5922)at a 1/200 dilution; anti-Oct4, a rabbit polyclonal antibody (SantaCruz, Catalog # sc-9081) at a 1/200 dilution; sheep anti-TyrosineHydroxlyase (TH) antibody (Pel-Freez, Catalog # P60101-0) at a 1/500dilution; anti-SSEA4, a mouse monoclonal antibody (Developmental StudiesHybridoma Bank, Catalog # MC-813-70) at a 1/5 dilution.

The cells were then washed in wash buffer (50 mM Tris-HCL pH 7.5, and2.5 mM NaCl; 3 times for 5 minutes each). The cells were then incubatedfor a minimum of 2 hours in secondary antibodies diluted 1:1000,followed by washing in wash buffer. The secondary antibodies wereAlexa-350 (blue), 488 (green) or 568 (red) conjugated goat anti-chicken,anti-rabbit, or anti-mouse antibodies, all available from MolecularProbes. The cells were stained with 5 ng/ml DAPI to detect cell nuclei,and were then washed from overnight to 2 days in a large volume of washbuffer. The slides were mounted with mounting medium and a cover slip.Slides were visualized using a either a NIKON TS100 inverted microscopeor a NIKON TE 2000-S inverted microscope with a Q Imaging digitalcamera.

Results

Human embryonic stem cell colony pieces grown in suspension culture inessentially serum free medium containing FGF-2, with or without MEDIIconditioned medium, became spherical within 6 hours of derivation, anddeveloped into essentially uniform spherical suspension cultures. Thepresence of MEDII appeared to be beneficial to overall morphology andcell viability during the early derivation of essentially serum freeembryoid bodies. sfEBMs exhibited reduced cell death in suspensionculture and improved overall morphology in comparison to sfEBs in thefirst three days after derivation. In general, sfEBM exhibited a fastergrowth rate than sfEBs.

After one week of culture, two distinct types of essentially serum freeembryoid bodies could be observed: a solid type, and a structured type.FIGS. 1A and 1B show derived serum free embryoid bodies. HESC colonieswere dissected into uniform sized pieces and cultured in suspension.Solid essentially serum free embryoid bodies (FIG. 1A) weredistinguished from structured essentially serum free embryoid bodies(FIG. 1B) after one week. Solid essentially serum free embryoid bodiesappeared highly uniform, and did not exhibit any obvious differentiationsuch as endoderm formation, or cavitation. Structured essentially serumfree embryoid bodies developed a morphology that appeared to becomprised of multiple spherical compartments, each of which may, or maynot, have contained an inner cavity. These appeared to be threedimensional spherical equivalents of the cell rosettes observed duringsubsequent adherent culture. Structured essentially serum free embryoidbodies also did not exhibit differentiation of an endoderm layer. Solidand structured embryoid bodies were observed with and without thepresence of MEDII. In some instances a single embryoid body exhibitedboth types of morphology as distinct regions.

To examine neural differentiation, essentially serum free embryoidbodies were seeded onto polyornithine/laminin coated slides and wereallowed to develop in adherent culture in essentially serum free medium.Both sfEBs and sfEBMs adhered, flattened somewhat and exhibitedoutgrowths of cells in culture periods of up to one week. The structuredsfEBs and sfEBMs could undergo proliferation and developed distinctivecellular organization to radial rosette structures, a characteristic ofneural stem/progenitor cells.

FIGS. 2A-2B show neural differentiation of serum free embryoid bodies.FIG. 2A shows rosette proliferation observed in sfEBs seeded in thepresence of 50% MEDII. FIG. 2B shows neuron network differentiationobserved in sfEBs seeded in the presence of 50% MEDII. Some of thestructured essentially serum free embryoid bodies developed outgrowthsthat appeared to be essentially pure populations of rosettes. Whileproliferation and development of rosettes were observed in the absenceof MEDII, they were far more extensive in the presence of MEDII. MEDIIthus has an obvious positive effect on the formation of neural celltypes.

Structured essentially serum free embryoid bodies exhibited extensivedifferentiation of neurons in the presence of MEDII, with large neuronnetworks observed. A markedly reduced level of neuron differentiationwas observed in adherent culture in the absence of MEDII. The structuredessentially serum free embryoid bodies contained rosettes consisting ofrelatively pure populations of neural precursors (Sox1⁺ cells), andpartially differentiated neural precursors (cells co-expressing Sox1 anda radial pattern of Map2 staining). These precursor cell types weretightly packed with nuclei distributed throughout the rosettes.Precursor cells were also closely associated with networks ofdifferentiated neurons (Map2⁺ cells) at their periphery. Rosette cellsalso co-expressed Nestin, a neural precursor marker, with the radialMap2 expression. The radial pattern of Map2 expression was clearlydifferent from the intense dendrite specific Map2 staining associatedwith differentiated and more mature neurites. Differentiated neuronswere observed in close association with or emanating from the rosettes,and were presumed to differentiate from these precursor cells.

Staining of the structured essentially serum free embryoid bodies withanti-Nestin, anti-Sox1, and anti-Map2 further demonstrated the presenceof multiple neural cells types: neural progenitors (Nestin⁺/Sox1⁺,and/or semi-differentiated neurons (Nestin⁺/Sox1⁺/radial Map2⁺),differentiated neurons (Map2⁺), and presumptive glial cells (Nestin⁺).FIGS. 3A-3C show serum free embryoid bodies containing neuralprecursors. FIG. 3A shows phase contrast micrograph of seeded structured(left) and solid (right) serum free embryoid bodies. Greyscale images offluorescent stainings with anti-Sox1 is shown in FIG. 3B and anti-Map2antibodies is shown in FIG. 3C. Solid serum free embryoid bodies containa high proportion of neural precursors (Sox1⁺ cells) and rare neurons(Map2⁺). Structured serum free embryoid bodies contain rosettes ofsemi-differentiated neural precursors (Sox1⁺/Map2⁺) and neurons (Map2⁺).

FIGS. 4A-4B show Map2 and nestin expression in rosettes. Radial Map2⁺and nestin⁺ expression indicates a semi-differentiated progenitor/youngneuron.

FIGS. 5A-5C show Sox1, Map2, and DAPI expression in serum free embryoidbody differentiation. Map2 expression in FIG. 5A indicates asemi-differentiated neuron (radial pattern) and differentiated neurons(network, dendritic stain), Sox1 expression in FIG. 5B indicates neuralprecursors, and DAPI in FIG. 5C is a DNA stain highlighting all nucleiin the field. Virtually all nuclei in the rosette are double stained forSox1/DAPI, indicating a relatively pure population of neuralprecursors/progenitors (Sox1⁺) and/or semi-differentiated neuralprecursors/progenitors (Sox1⁺/Map2⁺).

Solid essentially serum free embryoid bodies also exhibited extensiveproliferation, but did not develop rosettes or exhibit neurite networks.These outgrowth cultures were analyzed by immunocytochemistry for neuralmarkers. Staining with anti-Sox1 and anti-Map2 antibodies, whichidentify neural precursors and neurons respectively, demonstrated highlyefficient neural differentiation in these cultures. Solid essentiallyserum free embryoid bodies contained a high proportion of neuralprecursors (Sox1⁺ cells) and relatively few differentiated neurons(Map2⁺ cells).

Essentially serum free embryoid bodies could be maintained and werestable through multiple passages in suspension culture. MEDII appearedto enable more rapid proliferation of essentially serum free embryoidbodies over extended culture in suspension.

In an attempt to derive clones of essentially serum free embryoidbodies, trypsin was used to generate single cell suspensions from sfEBMcultures and low density cell suspensions, from 0-0.5-5 cells/μl, wereincubated in suspension dishes. Small uniform colonies were observed at4-5 days in essentially serum free medium that contained FGF-2 (4 ng/ml)and 50% MEDII. No clonal colonies could be generated in medium thatcontained FGF-2 or MEDII alone. The synergistic activities of FGF2 andMEDII in the derivation of clonal sfEBMs was unexpected. Titration ofthe MEDII revealed that this portion of the colony derivation activitycould function when diluted down to 10% of the medium. The coloniesproduced were presumed to be clonal, and proliferated to as much as 0.2mm in diameter within 10 days. The cloning efficiency was in the orderof 4.5-6%.

Cloned essentially serum free embryoid bodies passage 1 (sfEBMc1) weresimilar to solid essentially serum free embryoid bodies in overallmorphology, although the sfEBMc1s were highly sticky, and were able toadhere to and develop colonies on agarose coated plates. After a week inculture, some colonies detached and developed in suspension andexhibited a morphology very similar to solid essentially serum freeembryoid bodies. When seeded on polyornithine/laminin coated slides,sfEBMc1 developed highly homogeneous outgrowths. The outgrowth cellsgrew as a radial monolayer and were not obviously neural precursors orneurites, but did exhibit many cellular extensions, and appeared to beglial-like in morphology. No rosettes were observed. Staining withanti-Map2 revealed that these colonies retained some capacity forneuronal differentiation, although this was markedly reduced whencompared to solid essentially serum free embryoid bodies, with only rareneurons or very small neuronal networks being observed. SfEBMc1 culturesdid not exhibit any colonies similar to structured essentially serumfree embryoid bodies.

Passaging from sfEBMc1 colonies to sfEBMc2 cultures was possible usingthe same trypsin based approach, although the overall proliferation rateof sfEBMc2 colonies appeared to be slower than in sfEBMc1 cultures.

Example 6

Method to Derive Neuralized Serum Free EBM from HESC Crater Colonies

The colony morphology of HESCs differed from the typical multilayered,domed colonies when HESCs were plated onto feeder cells that had beenfreshly plated. When plated on feeder cells that were 0-6 hours old, butnot on feeders that were 2 days old or older, typical HESC coloniesformed except that in the central region of the colony a “crater” wasobserved. These central cells formed a monolayer of uniform cells,within a ring of multilayered HESCs. This monolayer was in directcontact with the tissue culture plastic, or the extracellular matrixthat was left behind as the HESC colony had pushed out the underlyingfeeder layer. HESC colonies typically displace the underlying feederlayer as they seed and proliferate. Cells within the crater expressedthe pluripotent marker Oct-4, although apparently at a reduced levelcompared to the surrounding ring of HESCs, indicating that they are anovel, partially differentiated cell type derived from the HESCs. Thisapproach allowing the controlled development of crater HESC coloniesoccurred within 3 to 5 days and generated a uniform monolayer of centralcells, as opposed to stochastic differentiation proceeding over severalweeks and leading to a complex heterogeneous culture.

The differentiation capacity of the crater cells was tested in the serumfree embryoid body system. Crater cells were purified by removing thefeeder layer and HESC growing on their surface. Watchmakers forceps wereused to hold the feeder layer at the side of the culture dish, and liftthis layer and attached multilayered HESC from the dish. Thismanipulation peels the feeder layer and the multilayered parts of theHESC colonies off of the dish and leaves behind the cells that hadformed the crater. The monolayer crater cells were left attached to thedish. Glass needles were used to cut the crater monolayer to 50-200 cellsize pieces, and lift them from the dish. These pieces were grown insuspension culture in same essentially serum free conditions as inExample 4 (DMEM/F12/N2/FGF-2 (4 ng/ml)/with or without 50% MEDII). After4 to 5 days suspension culture, serum free EB formed in this wayexhibited structured regions morphologically similar to those seen inExample 5.

While structured regions formed in serum free medium containing FGF-2,the presence of 50% MEDII significantly enhanced the derivation andoverall appearance of the structured regions, such that some EBs werecomprised of more than 80% structured material. When crater derivedsfEBs or sfEBMs were seeded onto polyornithine/laminin coated slides,significantly neuralized cell cultures were observed. Embryoid bodiesseeded 3-5 days after derivation developed rosette containing cultureswhen cultured in essentially serum free medium containing FGF-2, with orwithout 50% MEDII. Embryoid bodies that had been derived in the presenceof MEDII were seeded in the presence of MEDII, while embryoid bodiesthat had been derived without MEDII were seeded without MEDII. WithoutMEDII, the proportion of rosettes observed was from around 10-30% of thecolony area. In the presence of MEDII, the proportion of rosettesobserved increased to around 50-80% of the colony area. Other undefinedcell types were usually present in both conditions, but comprised ahigher proportion of the culture when MEDII was not included in themedium.

When crater derived embryoid bodies were seeded after prolongedsuspension culture, more than one week after derivation, or afterseveral passages of the structured material and more than one monthculture, extensive networks of neurons were observed deriving fromcolonies. Immunocytochemical staining demonstrated that neuralprogenitors (Nestin⁺/Sox1⁺), and/or semi-differentiated neurons(Nestin⁺/Sox1⁺/radial Map2⁺), differentiated neurons (Map2⁺), andpresumptive glial cells (Nestin⁺) were present in these cultures andcorresponded to morphological observations of these cell types. Ingeneral, the proportion of cultures differentiating to rosettes andneurons was higher when embryoid bodies were derived from crater HESCcolonies compared to multilayered HESC colonies. The presence of 50%MEDII enhanced the generation and/or proliferation of structured regionsof embryoid bodies, and rosettes when seeded onto apolyornithine/laminin matrix.

Dopaminergic Differentiation

To examine the level of dopaminergic differentiation in the seeded serumfree embryoid body cultures derived from crater cells, fluorescentimmunocytochemical staining was performed using a sheep anti-TyrosineHydroxylase (TH) antibody (Pel-Freez, #P60101-0, 1:500 dilution).Isolated TH+ neurons and networks of TH+ neurons were observed in craterderived serum free EBM seeded colonies (FIG. 7) and Map2+/TH+ neuronswere observed (FIG. 8). FIG. 7 shows a network of anti-TyrosineHydroxylase stained neurons in a crater derived sfEBM plated onto apolyornithine/laminin matrix. FIGS. 8A-8B show co-expression of TyrosineHydroxylase (FIG. 8A) and Map2 (FIG. 8B) shown by fluorescentimmunostaining of neurons in a crater derived sfEBM plated onto apolyornithine/laminin matrix. FIGS. 9A-9B show crater derived serum freeembryoid bodies at day 7 in suspension in (FIG. 9A) DMEM/F12/N2/FGF-2and (FIG. 9B) DMEM/F12/N2/FGF-2/50% MEDII.

Example 7

Reduction in the Level of Oct4 Protein in Differentiated HESCs

The Oct4 transcription factor is a tightly regulated marker ofpluripotency in the mouse, and expression of Oct4 mRNA in human innercell mass and ES cultures has been confirmed (Hansis et al., 2000, Mol.Hum. Reprod. 6(11), 999-1004, and Reubinoff et al., Nature Biotech.2000, 18, 399-404). However, the restriction of Oct4 protein topluripotent cells in humans has not been examined thoroughly. Manuallypassaged HESC cultures containing domed or cratered colonies werestained with anti-Oct4 antibodies.

It was observed that the Oct4 protein is expressed at high levels inundifferentiated HESCs (FIG. 10A) and that levels of the Oct4 proteinare down-regulated following differentiation (FIG. 10B). An unexpectedcharacteristic of immunostaining in the culture systems analyzed wasthat differentiated human cells retained a reduced but detectable levelof Oct4. However, when seeded sfEBM cultures were fixed andimmunostained, a process that maintains the morphology of a culture, thedifference between the two types of Oct4 expression was clearlydistinguishable. High level Oct4 expression was only observed as brightnuclear staining in tightly packed but evenly spaced cells. Thereforeimmunostaining for Oct4 expression during neural differentiation inembryoid bodies was a suitable assay for the presence of residualcompartments of pluripotent cells.

To monitor the persistence of pluripotent cells during sfEBMdifferentiation, essentially serum free embryoid bodies were generatedfrom domed HESC colonies or monolayer crater ES cells. The sfEBMs weregrown in suspension for 3-7 days, seeded onto polyornithine/laminincoated chamber slides, cultured for 3-5 days in the same medium andfixed for immunostaining. The presence of residual nests of pluripotentcells was demonstrated by clusters of high level Oct4 immunostainingamongst the generalized low level of Oct4 staining seen in theneuralized culture (FIG. 10C). The Oct4 immunoreactivity wasnuclear-specific. High level Oct4 expression was not associated with theneural rosettes, which were visualized by the characteristic radialpattern of nuclei stained with DAPI (FIG. 10D). The presence of nests ofresidual pluripotent cells was still observed in sfEBMs that werecultured for over one month, with several passages specificallyattempting to purify the neural rosette material, highlighting thepersistent nature of these pluripotent cells and their implied teratomaforming potential when transplanted.

Example 8

SSEA4 Selection and Protease Passaging Techniques Generate a HomogeneousCell Population from ES Cells

Methods

Embryoid bodies were generated from SSEA4 selected and bulk passagedcells as described in Example 5.

Immunostaining

Immunostaining was performed as described in Example 4 for nestin andOct4.

For immunostaining with SSEA1, SSEA3, SSEA4, Tra1-60, and Tra1-81,samples were washed in block buffer (3% goat serum, 1% PVP in PBS) for30 minutes, and then were incubated with the appropriated dilution ofthe primary antibody, or combination of antibodies for 4-6 hours at roomtemperature. The primary antibodies used were anti-SSEA1, a mouse IgMantibody (Developmental Studies Hybridoma Bank, Catalog # MC-480),undiluted; anti-SSEA3, a rat IgM antibody (Developmental StudiesHybridoma Bank, Catalog # MC-631), undiluted; anti-SSEA4, a mouse IgG3antibody (Developmental Studies Hybridoma Bank, Catalog # MC-813-70),undiluted; anti-Tra-1-60 (a gift from Peter Andrews), undiluted; andanti-Tra-1-81, (a gift from Peter Andrews), undiluted. The cells werethen washed in wash buffer (PBS) 3 times for 5 minutes each. Theremainder of the immunostaining protocol was performed as described inExample 5.

Results

Sorted HESCs contained the expected pattern of marker expression forundifferentiated pluripotent cells: SSEA4⁺, Oct4⁺, Tra-1-60⁺, Tra-1-81⁺,SSEA3⁺, and SSEA1⁻ (FIG. 11). Unexpectedly, SSEA4 selected HESC alsoexpressed the neural progenitor marker Nestin (FIG. 12). Manuallypassaged HESC cultures are typically heterogeneous, demonstrated bycolonies that contained a ring of cells expressing nestin thatsurrounded the bulk of the colony which did not exhibit nestinexpression (FIGS. 12A, and 12B). In comparison, SSEA4 selected HESCsshowed uniform nestin expression (FIGS. 12C, and 12D). Nestin is aintermediate filament protein that has a distinct pattern in neuralprogenitor cells. Nestin staining in SSEA4 selected HESCs was organizedinto a uniformly distributed filamentous staining. The lack of nestinexpression in the bulk of manually passaged HESCs in contrast to theuniform nestin staining in SSEA4 selected HESCs indicated that this bulkpassaged population, while identical to manually passaged HESCs withregard to expression of markers of pluripotency, could be a downstreamcell population with some pre-neural stem cell gene expressioncharacteristics. However, nestin may not be a tightly restricted neuralprogenitor marker (see Kachinsky et al., 1994 Dev. Biol., 165(1):216-28;Wroblewski et al., 1996 Ann. NY Acad. Sci. 8(785):353-5; Wroblewski etal., 1997 Differentiation, 61(3):151-9; and Mokry and Nemecek 1998, ActaMedica, 41(2):73-80).

Example 9

MEDII Enhanced Differentiation of SSEA4 Selected ES Cells

The application of 50% MEDII to embryoid bodies derived from SSEA4selected bulk passaged cells improved the neural differentiationsignificantly (FIG. 13). Without MEDII, extensive TH⁺ networks werepresent, but the proportion of the culture that did not contain neuronsand was presumably a non-neural background cell type varied betweenapproximately 30 and 90%. In the presence of MEDII, a consistently highproportion of the culture contained TH⁺ neurons, with the background ofnon-neural regions that was negative for the neuronal markerβIII-Tubulin typically lower than 10%. It was not determined whether theeffect of MEDII induced more efficient neuralization or inhibited thegeneration of non-neural cell types. Furthermore, neurons growing in thepresence of MEDII exhibited much longer cellular extensions and theyappeared more developed and differentiated than neurons in culturesexposed to FGF2 alone. Under this differentiation scheme, a very highproportion of all neurons, greater than 90%, expressed TyrosineHydroxylase (TH), the rate limiting enzyme in dopamine biosynthesis andthe standard marker for dopaminergic differentiation. This proportionwas determined by analysis of double staining of neural extensions forβIII-Tubulin and TH (FIG. 14), and overlaying Hoffman images with THimmunofluorescence (FIG. 15). The increase in the proportion of TH⁺neurons in MEDII treated differentiations appeared to be due to theoverall increase in neuronal differentiation, rather than an effect onthe proportion of neurons that were dopaminergic, because theproportions of neurons that were TH⁺ in differentiations not exposed toMEDII was equally high. Another marker of DA cells, VMAT, was expressedin similarly high proportions of cells within the sfEBM cultures.TH⁺/VMAT⁻, TH⁻/VMAT⁺ and TH⁺/VMAT⁺ cells were observed (FIG. 16),possibly indicating temporal variability in the induction of expressionof these markers prior to being co-expressed.

Example 10

Dopamine Release Assays Using sfEBM Cultures

Methods

Dopamine released by depolarized neural cultures was detected by using aCatecholamine-Enzyme Immunoassay (Labor Diagnostika Nord), a clinicaldiagnostic kit for determination of Dopamine in Plasma and Urine,according to the manufacturer's instructions. The experimental samplewas comprised of sfEBMs that had been derived, seeded topolyornithine/laminin coated slides at day 25 and cultured to day 30.Cells were depolarised by exposure to 300 μl 56 mM KCl in minimal MEM(Gibco) per well, for 15 minutes. The medium was removed and frozen.

The dopamine assay was performed as follows: (A) Dopamine was firstextracted from the sample using a cis-diol-specific affinity gel,followed by acylation to N-acyldopamine. The supplied standards and 300μl test sample were pipetted into wells of the cis-diol-specificaffinity gel coated plate. 50 μl assay buffer containing 1 M HCl wasadded to the wells, followed by 50 μl extraction buffer. The plate wascovered and incubated for 30 minutes at RT on an orbital shaker (600rpm). The liquid was decanted, 1 ml wash solution added and the platewas shaken for 5 minutes at 600 rpm. The liquid was decanted and thewash repeated. 150 μl acylation buffer, then 25 μl acylation reagent wasadded to the wells, followed by shaking at RT for 15 minutes at 600 rpm.The liquid was decanted and 1 ml wash solution added to wells, followedby shaking for 10 minutes at RT at 600 rpm. The liquid was decanted and150 μl 0.025 M HCl was added to wells to elute N-acyldopamine. 20 μl ofthe supernatant was used for the determination of dopamine. (B) TheN-acyldopamine was converted enzymatically to N-acyl-3-methoxytyaminefollowed by a competitive Dopamine-EIA. Acylated dopamine in suspensioncompetes with dopamine attached to the solid phase of a microtiter platefor a limited number of antiserum anti-dopamine binding sites untilequilibrium is reached. Free antigen and antibody complexes are removedby washing, and antibody complexed with the solid phase dopamine isdetected using a secondary antibody conjugated with peroxidase, usingTMB as a substrate and detected at 450 nm. The amount of antibody boundto the solid phase is inversely proportional to the dopamineconcentration of the sample.

The enzyme solution, catechol-O-methlytransferase, was made no longerthan 15 minutes prior to use, and was prepared by reconstitution with 1ml distilled water, followed by adding 0.3 ml Coenzyme,S-adenosly-L-methionine, and 0.7 ml Enzyme buffer. 25 μl of the enzymesolution was pipetted to assay wells, followed by 125 μl of 0.025 M HClinto the wells for the standards and controls. 10 μl of the extractedstandards, controls, two supplied patient urine samples and 125 μl ofthe extracted sfEBM sample was added to the appropriate wells followedby incubation at 37° C. for 30 minutes. 50 μl anti-dopamine antiserumwas added to all wells and shaken at room temperature for 2 hours at 400rpm. The wells were aspirated and washed twice with 300 μl wash bufferper well. 100 μl secondary antibody enzyme conjugate was added to thewells and shaken for 30 minutes at room temperature at 400 rpm. Thewells were aspirated and washed 3 times. 100 μl substrate was added toeach well and shaken for 35 minutes at room temperature at 400 rpm inthe dark. 100 μl stop solution was added to each well and the absorbancea 450 nm was read within 10 minutes. The absorbance for each standard,control and sfEBM sample were normalized for dilution and were plottedwith the linear absorbance of the standards along the y-axis versus logof the standard concentrations in pg/ml along the x-axis.

Results

sfEBM cultures were tested for the production and release of dopamine inresponse to KCl, a depolarizing agent. Cultures were treated with 56 μMKCl for 15 minutes and the culture supernatant assayed for the presenceof dopamine using a specific competitive ELISA. A seeded sfEBM culturesupernatant contained approximately 2657 pg/ml dopamine afterdepolarization (FIG. 17B), indicating that dopamine was synthesized bycells within the culture and released when treated with KCl. This valuedoes not indicate the absolute level of dopamine produced, as dopaminelevels would be affected by the number of dopaminergic cells seeded asembryoid bodies, their relative level of differentiation with regard todopamine biosynthetic pathways and vesicle production, and the volumeand subsequent dilution of the KCl supernatant. However, this value wassimilar to the 600 pg/ml found for cultures containing mouse DA neurons(Kim et al., 2002 Nature 418: 50-56), and it also fell between twounknown control samples supplied with the kit, although these values arenot directly comparable due to the above reasons.

Example 11

Differentiation of SSEA4 Selected HESCs in the Presence of Proline

To test their neural differentiation capacity in the presence ofproline, SSEA4 selected HESCs were differentiated in essentially serumfree conditions as embryoid bodies.

Methods

Essentially serum free embryoid bodies were generated from bulk passagedmonolayer HESC colonies as described in Example 5, in the presence of 4ng/ml FGF2 and 100 μM Proline, or in 4 ng/ml FGF2 with MEDII conditionedmedium as a positive control.

Serum free embryoid bodies were cultured in suspension for 17 days, andwere cut into pieces and seeded onto polyornithine/laminin coated slidesat day 10 or 17. The explants were cultured on slides for 5 days priorto fixation at day 15 or 22, for immunostaining with anti-βIII-Tubulinand anti-Tyrosine Hydroxylase antibodies.

Results

Serum free embryoid bodies grown in FGF2 and 100 μM proline (sfEBP)differentiated to neurons as observed by morphological andimmunofluorescent staining of seeded pieces (FIG. 18). Dense networks ofβIII-Tubulin⁺ cells were observed in the majority of seeded pieces(FIGS. 18A, and 18B). A proportion of seeded EB pieces, less than 30%,did not exhibit large networks of βIII-Tubulin⁺ cells and couldrepresent undifferentiated neural precursors, other neural cell types,or non-neural cells. Double immunofluorescent staining indicated thatgreater than 90% of the neurons generated were dopaminergic,co-expressing βIII-Tubulin and TH (FIGS. 18C, D, and E). This level ofdopaminergic differentiation was consistent with that observed with bulkpassaged SSEA4 selected HESCs differentiated in the presence ofFGF2/MEDII. Unlike sfEBMs, sfEBPs did not flatten when pieces wereseeded, and generally remained in a more globular structure. As notedpreviously, sfEBMs exhibit large outgrowths of a monolayer cell type(s),which neurons and neural extensions grew on top of. Therefore, sfEBMcultures exhibited long neuron extensions radiating from seeded pieces,which was not as pronounced in sfEBP pieces. Therefore the effect ofproline on the neural differentiation was pronounced, but did not mimicall the effects of MEDII. However, it is not clear if the proliferationof the monolayer cell type(s) will be beneficial for celltransplantations, and could effectively lower the proportions of neuronswithin the total culture, despite it being beneficial for in vitrodifferentiation of neural processes.

Example 12

Differentiation of SSEA4 Selected HESCs in Differing Media forFormulations

To test their neural differentiation capacity in the presence ofdifferent media formulations, SSEA4 selected HESCs were differentiatedin essentially serum free conditions as embryoid bodies.

Methods

Essentially serum free embryoid bodies were generated from bulk passagedmonolayer HESC colonies as described in Example 5, in the followingmedia formulations: βIII-Tubulin TH positive Media Formulation positivecells cells A minimal medium (DMEM, Not Not N2, L-Glutamine, determineddetermined Penicillin, Streptomycin) B minimal medium with 24% Not 4ng/ml FGF2 determined C minimal medium with 73% 51% 100 μM Proline Dminimal medium with 63% 60% 200 μM Proline E minimal medium with 31% 58%100 μM Proline and 4 ng/ml FGF2 F minimal medium with 36% 37% 200 μMProline and 4 ng/ml FGF2 G DMEM, F12, N2, L-Glutamine, 50% 52%Penicillin, Streptomycin and 4 ng/ml FGF2 H DMEM, F12, N2, L-Glutamine,25% 32% Penicillin, Streptomycin, 4 ng/ml FGF2 and 50% MEDII

Serum free embryoid bodies were cultured in suspension for 3 weeks.Morphological differences were apparent between the cultures. Lowproliferation in minimal medium (A) was observed, as well as increasedcell death, with an external layer of cell death surrounding whatappeared to be a viable and proliferative core of cells. Minimal mediumwith proline (C, D) seemed to exhibit a higher proliferation or survivalrate, although still contained increased cell death compared to FGF2containing conditions (B, E-H). Conditions B-H showed good proliferationover the course of the experiment. Serum free embryoid bodies werecultured in suspension, and were cut into pieces, seeded ontopolyornithine/laminin coated slides at day 21 and fixed at day 25.Immunostaining with anti-βIII-Tubulin demonstrated the presence ofextensive networks of neurons in all conditions, even in minimal medium(Condition A) that contained no FGF2, Proline, F12, or MEDII (FIG. 19).This was indicative that this differentiation protocol utilizes anintrinsic neural differentiation capacity of HESC, rather than exogenousneural inducing factors.

Cytospins of disaggregated serum free embryoid bodies were performed atday 21 to enable the counting of the proportion of βIII-Tubulin or THpositive cells generated in the different media formulations.βIII-Tubulin is a marker for differentiating neurons, but also known tobe expressed in HESC colonies, although this expression is notneuronal-like (Carpenter et al., Exp. Neurol. 172, 383-397). Expressionof βIII-Tubulin in seeded serum free embryoid bodies (FIGS. 18B, D; andFIG. 19), and in whole mount stainings of sfEBPs in suspension (FIG.20A), was only observed in cells of overt neuronal morphology.Therefore, using this marker to count the proportion of neurons insfEBPs is not expected to be influenced by the potential persistence ofpluripotent cells. The immunostaining of these cytospins with an anti-THantibody did not generate as strong a signal, and was therefore notlikely to be as accurate as the βIII-Tubulin count.

To count proportions of neurons in serum free embryoid bodies, cytospinswere immunostained with anti-βIII-Tubulin (Sigma, #T8660) or mouseanti-TH monoclonal antibodies (PelFreez Biologicals, #P80101-0),detected with alexa-488 conjugated anti-mouse secondary antibody andnuclei were stained with DAPI. Two color fluorescent images were takenunder 10× magnification and merged, and double positive signals werescored as neuronal cell bodies, or TH⁺ neuronal cell bodies against thetotal nuclei count. A minimum of three randomly sampled fields and 250or 100 nuclei for βIII-Tubulin or TH, respectively, were counted foreach condition. The highest proportion of βIII-Tubulin positive cellswas observed in L-Proline conditions (Conditions C and D), indicatingthe purest population of neurons generated in this comparison. Therelatively lower proportion of neurons observed in FGF2/MEDII conditions(Condition H, 25%) indicated the overgrowth of the presumptive glial orglial progenitor monolayer cell type observed morphologically, ratherthan a reduced total number of neurons. The presence of a lowerproportion of neurons in any condition containing FGF2 (Conditions B,E-H) presumably reflected the known activity of this factor inmaintaining undifferentiated neural progenitors (Okabe et al., Mech Dev.1996: 59(1):89-102).

This data indicated that neuronal differentiation occurred insuspension, and sfEBPs in particular were likely to be a mix of neuralprecursors and differentiating neurons. L-Proline media (Conditions Cand D) appeared to exhibit the purest population of neurons, at morethan 50% of the cells in a sfEBP, but it was not determined if thesecells were as differentiated as observed previously in seeded sfEBM,where there are non-neuronal cell types for neurites to grow on. Whereanalyzed, immunostaining of cytospins with anti-TH also revealed similarproportion of TH⁺ neurons in each condition as total neurons, given thecaveat of the lower confidence of the accuracy of the count. Regardless,counting of TH⁺ cell bodies indicated that the large majority of neuronsin all the conditions tested were TH⁺. It is likely that this analysiswill be improved as the cytospin immunostain assay for TH is optimizedfurther. An example of this would be to develop a triple stain assay forTH/βIII-Tubulin/DAPI.

The differentiation of βIII-Tubulin positive neurons in all theconditions, including minimal chemically defined medium (Condition A),indicated that this system was based on the intrinsic capacity of HESCto differentiate to neurons, rather than the addition of exogenous“neural inducing” factors. In this scenario, the activities ofL-proline, FGF2 and MEDII could be related to the proliferation andsurvival of cell types generated intrinsically within the system.Alternatively, components of the N2 supplement (insulin, transferrin,progesterone, selenite and putrescine) could effect a neural inducingactivity. However, these components, apart for transferrin, were testedand shown to not play a significant role in neural specification in amonolayer system of mouse ES cell differentiation (Ying et al., 2003Nat. Biotech. 21:183-186).

Example 13

Differentiation of SSEA4 Selected HESCs in Various Concentrations ofL-Proline

To test their neural differentiation capacity in the presence of a rangeof L-Proline concentrations, SSEA4 selected HESCs were differentiated inessentially serum free conditions as embryoid bodies.

Methods

Essentially serum free embryoid bodies were generated from bulk passagedmonolayer HESC colonies as described in Example 5, in the presence ofthe media set out below. Media Formulations A Minimal medium (DMEM, N2,L-Glutamine, Penicillin, Streptomycin) B Minimal medium with 5 μMProline C Minimal medium with 50 μM Proline D Minimal medium with 100 μMProline E Minimal medium with 500 μM Proline

Essentially serum free embryoid bodies formed in the presence of prolinecontaining medium are termed sfEBPs. sfEBPs were cultured in suspensionfor three weeks, and were passaged by manual cutting at around the 2week mark. sfEBPs exhibited a high level of cell death throughout thefirst 3 weeks of suspension culture, with an outer layer of dead cellsand generally slow proliferation when compared to EB formation inFGF2/MEDII conditions in previous experiments. At around 3 weeks, sfEBPsexhibiting low cell death and distinct neural rosette structures/foldswere observed in all conditions. The appearance of this type of sfEBPwas noticeably enhanced in the 50 μM Proline condition. A higherproportion of the sfEBPs exhibited this morphology in the 50 μM Prolinecondition than in other conditions, and their morphology was superior,with fewer associated dead cells and more noticeable neural rosettestructures.

sfEBPs derived in 50 μM L-Proline have been passaged and maintained in aproliferative state in suspension culture for more than 7 weeks afterinitial derivation and 3-4 weeks after proliferation of neural rosettestructures. This indicates that under these conditions there is abalance between rosette proliferation and neuronal differentiation. Whenseeded to polyornithine/laminin, a high proportion of DA differentiationwas still exhibited. When seeded in 50 μM L-Proline, a high degree ofcell death was observed in outgrowths, although good networks ofβIII-Tubulin⁺ neurons were still viable. When seeded in FGF2/MEDIImedium, morphologically healthy outgrowths were observed to containneurons and cells similar to the presumed glial or glial progenitorderived from rosettes. This indicated that there were cell types withinthe sfEBPs that were continuously generated that could not survive inthe minimal conditions. It is likely that this indicated that thesecells were differentiated from rosette cells.

sfEBPs grown in 50 μM L-Proline were fixed in suspension andimmuonstained with anti-βIII-Tubulin or DAPI in a wholemount assay.These sfEBPs were mounted and optically sectioned using a Leica TCS SP2Spectral Confocal Microscope. Networks of βIII-Tubulin⁺ neurons werevisualized throughout the sfEBP, as were DAPI stained neural rosettes(FIGS. 20A and B).

The high degree of cell death observed over the first 3 weeks is likelyto be indicative of the continual generation of cell types that are notviable under these serum- and serum replacer-free conditions, until thegeneration, maturation, or adaptation of a neural rosette cell that canproliferate in minimal medium, which is enhanced in the presence ofL-proline.

Example 14

Derivation and Characterization of a Neurosphere Population from HumanEmbryonic Stem Cells

In this example, the method illustrated in Example 3 was essentiallyrepeated utilising human ES cells, with the following differences. Forhuman ES cells the MEDII conditioning was conducted using the Filtrate(<10 Kda fraction) of serum-free MEDII. In addition, human cellaggregates were formed as suspension bodies in 50% serum-free MEDIIFiltrate for a period of up to 15 days with no change in media at EBM⁹.Neurospheres were then formed from embryoid bodies after disaggregationto near single cells.

Culture and Passage of Human ES Cells

SSEA4 selection of Human ES cells was carried out using magnetic beadseparation and these initially sorted cells have been used in the bulkpassaging protocol for these experiments as described in Example 5.

SF MEDII/Filtrate Preparation

MEDII conditioned medium was prepared as described in WO 99/53021. Thefiltrate fraction of MEDII was prepared by ultrafiltration through a 10⁴M_(r) cut-off membrane (Centricon-3 unit; Amicon) as described in WO99/53021. Essentially the filtrate contained molecules less than 10⁴M_(r).

Formation of Human Embryoid Bodies

Collagenase/trypsin passaged ES cells were prepared as a single cellsuspension and seeded at a density of 150 cells/μl in low attachment TCdishes (Costar). Cell aggregates were split 1:3 at day 2 and possibly atday 3 if required. Cultures were feed daily for 9 days and on day 9bodies were transferred to poly-L-ornithine/laminin coated 24 well traysin 0.5 ml of medium if adhesive culture was to be conducted. Another 0.5ml media was added to each well after 24 hours incubation. Adheredcultures or suspension cultures were fed daily for a further 8 days.

Adhesive Culture for Neural Differentiation

Embryoid bodies or neurospheres/aggregates are allowed to settle onto acoated surface to allow differentiation to occur (4 to 8 days). Thecoating can be on a plastic surface in either a tray or a coatedcoverslip.

Poly-L-Ornithine/Laminin Coating

300 μl of poly-L-ornithine 0.01% solution (Sigma Cat # P4957) was addeddirectly from bottle into each well of a 24 well tray or a 4 well tray.Trays were sealed with parafilm and incubated overnight at 4° C. Wellswere rinsed 3× with sterile MQ water. Laminin (Sigma Cat# L20-20) wasdiluted from a 1 mg/ml frozen stock to 1 μg/ml in sterile MQ water. 300μl of laminin (1 μg/ml) was added to each well. Trays were sealed withparafilm and incubated overnight at 4° C. Wells were rinsed 3× withsterile MQ water and then once with 1×PBS. Trays were stored with PBS at4° C. for up to 2 to 3 weeks. Prior to seeding wells were rinsed with 1×medium by adding 1 ml of seeding media and incubating at 37° C., 5% CO₂to equilibrate.

Preparation of Human Neurospheres

Trypsin-EGTA Disaggregation of Embryoid Bodies: a 10 ml pipette was usedto transfer bodies to a yellow capped tube. Media was aspirated and 5 mlSigma PBS added. Bodies were allowed to settle and the PBS was aspiratedand 1.25 ml of EGTA (pH 7.5) was added to the tube and bodies weresoaked for 5 minutes at room temperature. Solution was aspirated and 0.5ml trypsin was added to bodies for 30 seconds. Disaggregation of thebodies was carried out by gently pipetting them up and down with a P1000Gilson pipette until there are no large cell clumps. 0.5 ml FBS was thenadded and the disaggregation continued until solution was uniformlydispersed. 10 ml DMEM+5% FBS was then added and cells were spun at 300rpm for 1 minute to remove clumps. The supernatant was transferred intoa fresh yellow capped tube 15 ml conical bottom tube and cells pelletedat 1200 rpm for 4 minutes. The cell pellet was then resuspended in 100μl of DMEM+5% FBS and a count of viable cells was performed. Dissociatedcells were then seeded into a T25 flask @ 50-100 cells/μl in 6 mls ofneurosphere media (NSM; DMEM/F12, B27 1:50, ITSS 1:100, Heparin (10mg/ml) 1:1000, FGF2 ((25 mg/ml) 1:5000 dilution) and spheres allowed toform over a two-three week period. NSM was changed 50:50 every 4 days.

Passaging of Neurospheres

Disaggregation of neurospheres was conducted either using the trypsindissociation method described above for the preparation of neurospheresor using a mechanical trituration method as follows. Using a 10 mlpipette, spheres were transferred to a 15 ml yellow capped conicalbottom tube. Spheres that had attached to the flask were gentlydislodged with 5 mls fresh media and added to the tube. Spheres werepelleted by centrifugation. The supernatant was removed, leaving behindapproximately 200 uL and the pellet gently triturated approximately 150×using a p200 pipetteman. 5 ml of culture medium was added andcentrifuged gently to remove debris. The supernatant was removed andcells were gently dissociated 10-20× to disaggregate the pellet. Aviable cell count was done and cells were reseeded at 1×10³ cells/cm²(equivalent to ˜4 cells/μl).

Results

In the presence of MEDII filtrate, neurospheres were derived from EBM⁹s.If filtrate was omitted, derivation of neurospheres from EBMs wasdelayed until EBM¹²⁻¹⁵.

Neurospheres contained neuronal cells (NF200+ve). Neurospheres alsoincluded glial cells (GFAP⁺). TH⁺ neurons were also present afterpassaging.

Example 15

Derivation and Characterization of a Neurosphere Population from HumanEmbryonic Stem Cells Using a Two-Stage Process

Essentially, the process using mouse ES cells, as outlined in Example 14was repeated with some modifications using human ES cells. Human ES cellculture, cell aggregate/embryoid body formation and adherent culture wasessentially as described in Example 14.

Basic Media (DMEM/F12 and ITSS or N2).

Embryoid bodies/neurospheres from human ES cells were grown without theuse of MEDII conditioned media Media and supplements used were HamsDMEM/F12 (Gibco Cat # 11320-033), ITSS (Gibco Cat#17502-048) and N2(Gibco Cat#41400-045). The media did not contain HEPES.

Comparison of Media

The ability of basic media with supplements (DMEM+N2 or ITSS) to promoteneural differentiation of hES cells was compared with medium thatincluded F12: (DMEM:F12 (1:1)+N2 or ITSS).

Initial results showed that embryoid bodies can form in either of thesebasic media even without FGF2. Immunohistochemistry for NF200 revealedthat under both media conditions with either supplement, neurons formed.Furthermore, without the addition of a mitogen such as FGF2, there werestill proliferating, Ki67 positive cells. An important distinctionbetween the two media is that TH⁺ positive cells were present in largenumbers (˜50%) in DMEM/F12 and either supplement (N2 or ITSS), but notin DMEM only with either supplement.

Trypsinised Human ES cells were seeded at approximately 10 to 20cells/μl of media into 10 ml of neurosphere media (DMEM:F12, 10 μg/mlheparin (SIGMA), 1/50 B27 (GIBCO), 1/100 pen/strep, 1/100 ITSS) in a T75culture flask. 10 ng/ml FGF2 was optionally added but the culture mediumwas preferably mitogen-free (no FGF2). Cultures were maintained in themedia for 9 days after which the embryoid bodies were optionallytransferred to poly-L-ornithine/laminin plates and cultured in the samemedia for a further 6 days. The embryoid bodies so formed (EB¹⁵),whether from adherent or suspension culture, were then triturated tonear single cell form and used for either transplantation or for theformation of neurospheres/cell reaggregates.

Formation of neurospheres was achieved as described in Example 14. Theneurospheres formed were seeded onto poly-L-ornithine/laminin-coatedplates and allowed to adhere and differentiate. Optionally in thisculture stage, neurospheres can be maintained in media containingcombinations of RA, 50% MEDII or filtrate, and L-proline. In analternative treatment, during stage A, the embryoid bodies (EB⁹) can beseeded onto poly-L-ornithine/laminin-coated culture plates and culturedfor 6 to 8 days to permit neuronal differentiation.

In an alternative treatment (Stage B), neurosphere formation wasachieved when embryoid bodies formed from stage A were triturated andresuspended in a minimal media (DMEM/F12 and N2 or ITSS). Optionally,this media can also include combinations of FGF, MEDII, RA andL-proline. The aggregates formed can also be seeded ontopoly-L-ornithine/laminin coated plates and allowed to adhere anddifferentiate.

Results

In the presence of F12 media, embryoid bodies formed that when adheredand differentiated formed high numbers of TH⁺ cells. If F12 was omitted,very few TH⁺ cells were observed, however, many NF200 positive cellswere present, suggesting that the absence of TH⁺ neurons was not due toan inability of neuronal cells to differentiate under these growthconditions.

The presence of FGF2 appears to have had little impact on the generationof TH⁺ cells. However, there was a more extensive outgrowth of cellsaround the seeded body when FGF2 was present in the media.

The neurospheres contained NF200⁺ neuronal cells, GFAP⁺ glial cells andoligodendrocytes.

Example 16

Derivation and Characterization of a Neurosphere Population from HumanEmbryonic Stem Cells Using a Two-Stage Process and Minimal Media

Example 15 was repeated utilising human ES cells and a minimal mediaconsisting of DMEM and 100 μM L-proline. The results were similar tothose described in Example 5.

Human ES cell culture, embryoid body/cell aggregate formation, adherentculture and passaging to form neurospheres or cell reaggregates wereessentially conducted as outlined in Example 14.

L-Proline

EB¹⁷ bodies formed in medium that contained DMEM and 100 μM L-prolinewere comprised of proliferating Ki67⁺ cells, NF200⁺ neuronal cells, anda high proportion (˜50%) TH⁺ cells. When the medium excluded L-proline,the TH⁺ cell content of EB¹⁷ bodies was reduced significantly.Generation of EBs with high proportions of TH⁺ cells occurred in theabsence of FGF2. Cells grown in the DMEM and N2 or ITSS did not producea significant population of TH⁺ cells.

Formation of neurospheres was achieved as described in Example 14. Theneurospheres formed were seeded onto poly-L-ornithine/laminin coatedplates and allowed to adhere and differentiate. Optionally in thisculture stage, neurospheres are maintained in media containingcombinations of RA, 50% MEDII and L-Proline. In an alternativetreatment, during stage A, the embryoid bodies (EB⁹) are seeded ontopoly-L-ornithine/laminin coated culture plates and cultured for 6 to 8days to permit neuronal differentiation. The neurospheres were positivefor the neurofilament marker NF200, and included glial cells that wereGFAP⁺, as well as oligodendrocytes.

In an alternative treatment, embryoid bodies cultured from stage A aretriturated and resuspended in a minimal media (DMEM/F12 and N2 or ITSS).Optionally this media also includes combinations of FGF, MEDII, RA andL-Proline. The aggregates formed are seeded ontopoly-L-Ornithine/laminin coated plates and allowed to adhere anddifferentiate.

Example 17

Study of Cell Implants in Sprague-Dawley Rats

Example 14 was repeated with certain modifications. Single Human EScells (trypsinised) were grown in a standard suspension culturecontaining 50% MEDII filtrate in the presence of FGF2. At day 9 theembryoid bodies formed (EBM⁹) were transferred topoly-n-ornithine/laminin coated plates in the same serum-free MEDIIfiltrate culture medium, maintained for a further 8 days and allowed toadhere. The use of FGF2 in the serum-free MEDII filtrate culture mediumwas optional. The embryoid bodies so formed (EBM¹⁷) were thentrypsinised to near single cell form. A cell suspension of 100,000cells/μl was stereotaxically injected (100,000 cells/μl per animal) intothe 6-OHDA lesioned striatum of eight Sprague-Dawley rats. A group of 5Rats was also included that did not receive cell implants, and acted assham controls. Rats were maintained under conditions ofimmunosuppression using Cyclosporin A (10 mg/kg) for a period of 8 weeksand rotational data was collected. Grafted human cells were detectedusing a human Alu-repeat DNA detection system.

After the 8 week period, the 8 implanted rats showed a statisticallysignificant reduction in their rotational scores compared to the controlgroup, with Single Factor ANOVA, p=0.047.

Immunohistochemical characterization of the human cell implants revealedneural lineages such as glial cells, and low numbers of neural cellspositive for the dopaminergic neurone marker, Tyrosine Hydroxylase(TH⁺). Implanted human cells from one rat (N274) expressed the neuronalmarker GFAP an astrocyte/glial lineage marker, DAPI, a non-specificnuclear marker and an Alu DNA probe in situ specific for detection ofhuman cells, thereby showing that implanted human cells were able todifferentiate to glia. Other implanted human cells from another rat(N278) expressed the neural precursor marker Nestin, human specific AluDNA probe in situ, and a general nuclear marker DAPI.Immunohistochemistry was preformed on the implants to detect thepresence of TH⁺ cells using chromogens. At least one rat (N278)contained implanted cell that expressed the dopaminergic neurone lineagemarker Tyrosine Hydroxylase. A small cluster of TH⁺ cells weredetectable with clearly staining cell bodies.

Example 18

Study of Cell Implants in Sprague-Dawley Rats

Example 17 was repeated utilising minimal culture media (DMEM:F12, andITSS or N2) with or without 10 μg/ml FGF2 in both stages A and B. Thisproduced embryoid bodies at days 15 to 17 (EB 15 to 17) containing highnumbers of TH positive neuronal cells (see Example 15).

The cells were trypsinised to an essentially single cell suspension. 1μl containing approximately 100,000 cells, was transplanted into a ratmodel as described above. FGF2 was not included in the culture mediumused to prepare cells for transplant, however, inclusion of FGF2 in theculture medium is optional. In one embodiment, EB⁹s are cultured onlaminin/polylornithine coated plates for a further period of up to 8days.

Example 19

Identification of Neural Cells with Specific Markers

Neural cells produced according to the essentially serum free mediamethods as described above are identified by expression of detectablemarkers. The markers used in this example include tyrosine hydroxylase(TH), which is the rate limiting enzyme in dopamine biosynthesis.Another marker is the aromatic amino acid decarboxylase (AADC, alsoknown as dopa decarboxylase) which is the second enzyme in the pathwayfor dopamine synthesis. Also detected is vesicular monoamine transporter(VMAT), the vesicular transporter that packages dopamine (and othercatecholamines) into synaptic vesicles. Therefore, VMAT is required fordopamine release. Further detected is dopamine transporter (DAT), theplasma membrane transporter that brings dopamine back into the cellafter it has been released. DAT is likely the most specific marker fordopaminergic neurons. The co-expression of these markers within a neuronis required for the synthesis, vesicular packaging and reuptake ofdopamine, consistent with the normal function of a dopaminergic neuron.

For immunostaining, the neural cells are rinsed with 1×PBS and fixed in4% paraformaldehyde, 4% sucrose in 1×PBS for 30 minutes at 4° C. Thecells are then washed in 1×PBS and stored at 4° C. To performimmunostaining, the cells are washed in block buffer (3% goat serum, 1%polyvinyl Pyrolidone, 0.3% Triton X-100 in wash buffer) for 30 minutes,and then incubated with the appropriate dilution of the primaryantibody, or combination of antibodies for 4-6 hours at roomtemperature. The primary antibodies are anti-TH, a sheep monoclonalantibody (Pel-Freez, Catalog # P60101-0) at 1:500 dilution; anti-VMAT2,a rabbit monoclonal antibody (Chemicon, Catalog # AB1767) at a 1/500dilution; anti-DAT, a rabbit monoclonal antibody recognizingextracellular loop 2 of DAT (Chemicon, Catalog # AB5802) at a 1/100dilution; and anti-DAT, a rat monoclonal antibody recognizing anintracellular DAT epitope (Chemicon, Catalog # MAB369) at a 1/200dilution. For anti-AADC, for a primary antibody, one of a rabbitanti-dopa decarboxylase polyclonal antibody (Chemicon AB136 or ChemiconAB1569), and a sheep anti-dopa decarboxylase polyclonal antibody(Chemicon AB119) is used.

The cells are then washed in wash buffer (50 mM Tris-HCL pH 7.5, and 2.5mM NaCl; 3 times for 5 minutes each). The cells are then incubated for aminimum of 2 hours in secondary antibodies diluted 1:1000, followed bywashing in wash buffer. The secondary antibodies are Alexa-350 (blue),-488 (green) or -594 (red) conjugated goat anti-sheep, anti-rabbit, oranti-rat antibodies, all available from Molecular Probes. The cells canbe stained with 5 ng/ml DAPI to detect cell nuclei. The cells are thenwashed from overnight to 2 days in a large volume of wash buffer. Theslides are mounted with mounting medium and a cover slip. Slides arevisualized using a NIKON TE 2000-S inverted microscope or a NIKON E1000upright microscope with a Q Imaging digital camera.

Neural cells produced from HESCs under serum free conditions asdescribed herein are found to be TH⁺, VMAT2⁺, DAT⁺, and AADC⁺ byimmunostaining. This example demonstrates the successful in vivoproduction of a neural cell population from pluripotent HESC that isbelieved to be capable of dopaminergic production. Therefore, such aneural cell culture is expected to be a viable candidate for therapeutictransplantation to alleviate conditions characterized by dopaminergicdeficiency, such as Parkinson's disease.

Example 20

Identification of Neural Cells with Specific Markers

Neural cells produced according to the essentially serum free mediamethods as described above are identified by expression of detectablemarkers. The markers used in this example include nestin and vimentin.Preferably, the neural cells produced using the methods identifiedherein have the capacity to differentiate into cells of the neurallineage, including into neurons and glial cells. The neural cells typesproduced may include cells of the central or peripheral nervous systemincluding but not limited to neurons, astrocytes, oligodendrocytes andSchwann cells. Neuron cell types produced in these cultures may expressone or more neurotransmitter phentotypes. These include GABAergicneurons that express glutamate decarboxylase (GAD) or vesicularinhibitory amino acid transporter/vesicular gaba transporter(Viaat/Vgat); cholinergic neurons that express choline acetyltransferase(ChAT/CAT) or vesicular acetylcholine transporter (VAChT); glutamatergicneurons that express the vesicular glutamate transporter; glycinergicneurons that express the vesicular inhibitory amino acid transporter(Viaat/Vgat), noradrenergic neurons that express the norepinephrinetransporter (NET); adrenergic neurons that express phenylmethanolamineN-methyl transferase (PNMT); serotonergic neurons that expresstryptophan hydroxylase (TrH) or serotonin transporter (SERT); orhistaminergic neurons that express histidine decarboxylase (HDC).

For immunostaining, the neural cells are rinsed with 1×PBS and fixed in4% paraformaldehyde, 4% sucrose in 1×PBS for 30 minutes at 4° C. Thecells are then washed in 1×PBS and stored at 4° C. To performimmunostaining, the cells are washed in block buffer (3% goat serum, 1%polyvinyl Pyrolidone, 0.3% Triton X-100 in wash buffer) for 30 minutes,and then incubated with the appropriate dilution of the primaryantibody, or combination of antibodies for 4-6 hours at roomtemperature.

The cells are then washed in wash buffer (50 mM Tris-HCL pH 7.5, and 2.5mM NaCl; 3 times for 5 minutes each). The cells are then incubated for aminimum of 2 hours in secondary antibodies diluted 1:1000, followed bywashing in wash buffer. The secondary antibodies are Alexa-350 (blue),-488 (green) or -594 (red) conjugated goat anti-sheep, anti-rabbit, oranti-rat antibodies, all available from Molecular Probes. The cells canbe stained with 5 ng/ml DAPI to detect cell nuclei. The cells are thenwashed from overnight to 2 days in a large volume of wash buffer. Theslides are mounted with mounting medium and a cover slip. Slides arevisualized using a NIKON TE 2000-S inverted microscope or a NIKON E1000upright microscope with a Q Imaging digital camera.

This example demonstrates the successful in vivo production of a neuralcell population from pluripotent HESCs that is believed to be capable ofneurotransmitter production. Therefore, such a neural cell culture isexpected to be a viable candidate for therapeutic transplantation toalleviate conditions characterized by neurotransmitter deficiency.

1. A method of producing a human neural cell comprising, a) providing apluripotent human cell; b) forming an embryoid body by contacting thepluripotent human cell with an essentially serum free medium; and c)culturing the embryoid body in an essentially serum free celldifferentiation environment to thereby produce the human neural cell. 2.The method of claim 1, wherein the essentially serum free medium of stepb) comprises Ham's F12 medium.
 3. The method of claim 1, wherein theessentially serum free medium of step b) comprises a MEDII conditionedmedium.
 4. The method of claim 1, wherein the essentially serum freecell differentiation environment of step c) comprises a MEDIIconditioned medium.
 5. The method of claim 1, comprising the additionalsteps performed after step b) and before step c): a) dispersing theembryoid body to an essentially single cell suspension; b) culturing theessentially single cell suspension in a suspension culture; and c)forming a second embryoid body by culturing the essentially single cellsuspension with a second essentially serum free medium, wherein thesecond essentially serum free medium comprises a MEDII conditionedmedium.
 6. The method of claim 5, wherein one or more of the essentiallyserum free media is essentially LIF free.
 7. The method of claim 5,wherein the second essentially serum free medium comprises DMEM/F12,FGF-2 and a MEDII conditioned medium.
 8. The method of claim 5, whereinthe second essentially serum free medium comprises between approximately10% to approximately 50% MEDII conditioned medium.
 9. The method ofclaim 5, wherein the essentially serum free medium, the secondessentially serum free medium, and/or the essentially serum free celldifferentiation environment comprises less than 5% serum.
 10. The methodof claim 5, wherein the cell differentiation environment is selectedfrom the group consisting of adherent culture and suspension culture.11. The method of claim 1, wherein the human cell is a pluripotent humancell.
 12. The method of claim 11, wherein the pluripotent human cell isselected from the group consisting of a human embryonic stem cell, ahuman inner cell mass (ICM)/epiblast cell, a human primitive ectodermcell, a human primordial germ cell, a human teratocarcinoma cell. 13.(canceled)
 14. (canceled)
 15. The method of claim 12, wherein the humanprimitive ectoderm cell is an early primitive ectoderm (EPL) cell. 16.(canceled)
 17. (canceled)
 18. The method of claim 11, wherein thepluripotent human cell is a human embryonic stem cell, and wherein thehuman embryonic stem cell is passaged by selection with a SSEA4 antibodyand/or with a sequential collagenase and trypsin treatment prior toforming an embryoid body.
 19. The method of claim 1, wherein the humancell is a multipotent human cell.
 20. The method of claim 5, wherein theMEDII conditioned medium is a Hep G2 conditioned medium.
 21. (canceled)22. (canceled)
 23. (canceled)
 24. The method of claim 5, wherein theMEDII medium comprises a low molecular weight component selected fromthe group consisting of a proline residue, and a polypeptide comprisingone or more proline residues.
 25. (canceled)
 26. A neural cell producedby the methods of claim
 1. 27. A method for treating a patient,comprising a step of administering to the patient having a neuraldisease a therapeutically effective amount of the neural cell of claim26.
 28. The method of claim 27, wherein the neural disease isParkinson's disease.
 29. A method of producing a partiallydifferentiated pluripotent cell comprising culturing a pluripotent cellculture on a layer of fresh feeder cells, wherein the fresh feeder cellshave been plated for less than approximately 48 hours, thereby inducingformation of a more differentiated pluripotent cell.
 30. The method ofclaim 29, wherein the fresh feeder cells have been plated for less thanapproximately 24 hours.
 31. The method of claim 29, wherein the freshfeeder cells have been plated for less than approximately 12 hours. 32.The method of claim 29, wherein the fresh feeder cells have been platedfor less than approximately 6 hours.
 33. The method of claim 29, whereinthe pluripotent cell culture forms a colony after it is cultured on thelayer of fresh feeder cells, and the more differentiated pluripotentcell is selected from the central region of the colony.
 34. The methodof claim 33, wherein the more differentiated pluripotent cell expressesless Oct4 than an embryonic stem cell.
 35. The partially differentiatedcell generated using the method of claim
 29. 36. A neural cell culturecomposition comprising a population of neural cells derived in vitrofrom pluripotent cells, wherein the neural cells express one or moredetectable markers for tyrosine hydroxylase (TH), vesicular monaminetransporter 2 (VMAT2), aromatic amino acid decarboxylase (AADC) anddopamine transporter (DAT).
 37. (canceled)
 38. The composition of claim36, wherein at least one of the cultured cells expresses all of thedetectable markers TH, VMAT2, AADC and DAT.
 39. The composition of claim36, wherein the neural cell is a human cell.
 40. A neural cell culturecomposition comprising a population of neural cells derived in vitrofrom pluripotent cells, wherein the neural cells express one or moredetectable markers for nestin or vimentin, and the neural cells have thecapacity to differentiate into cells of a neural lineage.
 41. (canceled)42. The neural cell culture composition of claim 40, wherein the neurallineage is selected from the group consisting of neurons, astrocytes,oligodendrocytes and Schwann cells.
 43. (canceled)
 44. The neural cellculture composition of claim 40, wherein the cells of the neural lineageexpress a neurotransmitter phenotype selected from the group consistingof a GABAergic neuron, a cholinergic neuron, a glutamatergic neuron, aglycinergic neuron, a noradrenergic neuron, an adrenergic neuron, asertonergic neuron, and a histaminergic neuron.
 45. The neural cellculture composition of claim 44, wherein the neurotransmitter phenotypeis a GABAergic neuron that expresses glutamate decarboxylase and/orexpresses vesicular inhibitory amino acid transporter/vesicular gabatransporter.
 46. The neural cell culture composition of claim 44,wherein the neurotransmitter phenotype is a cholinergic neuron thatexpresses choline acetyltransferase and/or vesicular acetylcholinetransporter.
 47. The neural cell culture composition of claim 44,wherein the neurotransmitter phenotype is a glutamatergic neuron thatexpresses vesicular glutamate transporter.
 48. The neural cell culturecomposition of claim 44, wherein the neurotransmitter phenotype is aglycinergic neuron that expresses vesicular inhibitory amino acidtransporter.
 49. The neural cell culture composition of claim 44,wherein the neurotransmitter phenotype is a noradrenergic neuron thatexpresses norepinephrine transporter.
 50. The neural cell culturecomposition of claim 44, wherein the neurotransmitter phenotype is aadrenergic neuron that expresses phenylmethanolamine N-methyltransferase.
 51. The neural cell culture composition of claim 44,wherein the neurotransmitter phenotype is a serotonergic neuron thatexpresses tryptophan hydroxylase or serotonin transporter.
 52. Theneural cell culture composition of claim 44, wherein theneurotransmitter phenotype is a histaminergic neuron that expresseshistidine decarboxylase.
 53. The composition of claim 40, wherein theneural cell is a human cell.